Conjugate vaccine for group B streptococcus

ABSTRACT

A vaccine capable of protecting a recipient from infection caused by group B Streptococcus. The vaccine provides polysaccharide-protein moieties and contain (a) a group B Streptococcus polysaccharide conjugated to (b) a functional derivative of a group B Streptococcus C protein alpha antigen that retains the ability to elicit protective antibodies against group B Streptococcus. The vaccine may contain only one type of such polysaccharide-protein unit or may contain a mixture of more than one type of unit.

This invention was made with government support; the government hascertain rights in this invention.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a division of application Ser. No. 08/363,311, filedDec. 22, 1994, now U.S. Pat. No. 5,648,241 which is a continuation ofapplication Ser. No. 07/968,866, filed Nov. 2, 1992, now abandoned,which is a continuation-in-part of U.S. application Ser. No. 07/408,036,filed Sep. 15, 1989, now abandoned.

FIELD OF THE INVENTION

The invention relates to the fields of microbiology and vaccinetechnology, and concerns the development of a vaccine capable ofconferring immunity to infection by group B Streptococcus.

BACKGROUND OF THE INVENTION

Bacteria of the Streptococcus genus have been implicated as causalagents of disease in humans and animals. The Streptococci have beendivided into immunological groups based upon the presence of specificcarbohydrate antigens on their cell surfaces. At present, groups Athrough O are recognized (Davis, B. D. et al., In: Microbiology, 3rd.Edition, page 609, (Harper & Row, 1980). Streptococci are among the mostcommon and important bacteria causing human disease. AlthoughStreptococci of the B group are associated with animal disease (such asmastitis-in cattle), Streptococcus agalactiae (a group B Streptococd)has emerged as the most common cause of human neonatal sepsis in theUnited States and is thought to be responsible for over 6000 deathsannually (Hill, H. R. et al., Sexually Transmitted Diseases, McGrawHill, pp. 397-407). Group B Streptococcus is also an important pathogenin late-onset meningitis in infants, in postpartum endometritis, and ininfections in immunocompromised adults (Patterson, M. J. et al., Bact.Rev. 40:774-792 (1976)). Although the organism is sensitive toantibiotics, the high attack rate and rapid onset of sepsis in neonatesand meningitis in infants results in both high morbidity (50%) andmortality (20%) (Baker, C. J. et al., New Eng. J. Med. (Editorial)314(26):1702-1704 (1986); Baker, C. J. et al., J. Infect. Dis.136:137-152 (1977)).

Group B Streptococcus is a common component of normal human vaginal andcolonic flora. While the most common route of neonatal infection isintrapartum from vaginal colonization, nosocomial spread in newbornnurseries has also been described (Patterson, M. J. et al., Bact. Rev.40:774-792 (1976)). However, only a small percentage of infantscolonized with group B Streptococcus develop serious infections. Therole of both host factors and bacterial virulence determinants in thetransition from colonization to infection is not well understood.

Several proteins from group B Streptococcus are thought to have a rolein virulence and immunity (Ferrieri, P, Rev. Infect. Dis. 10:S363(1988)). In 1975, Lancefield defined the C proteins of group BStreptococcus by their ability to elicit protective immunity(Lancefield, R. C, et at., J. Exp. Med. 142:165-179 (1975)). This groupof proteins is thought to contain several different polypeptides andantigenic determinants. In view of these findings, efforts to preventinfections with group B Streptococcus have been directed towards the useof prophylactic antibiotics and the development of a vaccine againstgroup B Streptococcus (Baker, C. J, et al., Rev. of Infec. Dis.7:458-467 (1985), Baker, C. J. et al., New Eng. J. Med. (Editorial)314(26):1702-1704 (1986)). Polysaccharide vaccines against group BStreptococcus are described by Kasper, D. L. (U.S. Pat. No. 4,207,414and U.S. Pat. No. RE31672, and U.S. Pat. Nos. 4,324,887, 4,356,263,4,367,221, 4,367,222, and 4,367,223), by Carlo, D. J. (U.S. Pat. No.4,413,057, European Patent Publication 38,265), and by Yavordios, D. etal (European Patent Publication 71,515), all of which references areincorporated herein by reference.

Except for the small sub-population of infants in whom both maternalcolonization with group B Streptococcus and other perinatal risk factorscan be identified, the use of prophylactic antibiotics has not beenpractical or efficacious in preventing the majority of cases (Boyer, K.M, et al., New Eng. J. Med. 314(26):1665-1669 (1986)). Intrapattumchemoprophylaxis has not gained wide acceptance for the followingreasons: (1) It has not been possible to identify maternal colonizationby group B Streptococcus in a fast, reliable and cost-effective manner;(2) About 40% of neonatal cases occur in low-risk settings; (3) It hasnot been considered practical to screen and/or treat all mothers orinfants who are potentially at risk; and (4) antibiotic prophylaxis hasnot appeared to be feasible in preventing late-onset meningitis (7200cases per year in the United States) or postpartum endometritis (45,000cases annually) (Baker, C. J. et al., New Eng. J. Med. (Editorial)314:1702-1704 (1986)).

DEPOSIT OF MICROORGANISMS

Plasmids pJMS1 and pJMS23 are derivatives of plasmid pUX12 which containDNA capable of encoding antigenic Streptococci proteins that may be usedin accordance with the present invention. Plasmid pUX12 is a derivativeof plasmid pUC12. Plasmids pJMS1 and pJMS23 were deposited on Sep. 15,1989, at the American Type Culture Collection, Rockville, Md. and giventhe designations ATCC 40659 and ATCC 40660, respectively.

SUMMARY OF THE INVENTION

Streptococcus agalactiae is the most common cause of neonatal sepsis inthe United States and is responsible for between 6,000 and 10,000 deathsper year. While the type-specific polysaccharide capsule of group BStreptococcus is immunogenic and carries important protective antigens,clinical trials of a polysaccharide vaccine have shown a poor responserate (Baker, C. J. et al., New Engl. J. Med. 319:1180 (1980); Insel, R.A, et al., New Eng. J. Med. (Editorial) 319(18):1219-1220 (1988)).

The present invention concerns the development of a conjugate vaccine togroup B Streptococcus, (i.e. Streptococcus agalactiae) that utilizes toa protective protein antigen expressed from a gene cloned from group BStreptococcus. This novel conjugate vaccine has the advantages both ofeliciting T-cell dependent protection via the adjuvant action of thecarrier protein and also providing additional protective epitopes thatare present on the cloned group B Streptococcus protein (Insel, R. A, etal., New Eng. J. Med. (Editorial) 319(18):1219-1220 (1988); Baker, C. J,et al., Rev. of Infec. Dis. 7:458-467 (1985)).

In detail, the invention provides a conjugate vaccine capable ofconferring host immunity to an infection by group B Streptococcus whichcomprises (a) a polysaccharide conjugated to (b) a protein; wherein boththe polysaccharide and the protein are characteristic molecules of thegroup B Streptococcus, and wherein the protein is a derivative of the Cprotein alpha antigen that retains the ability to elicit protectiveantibodies against the group B Streptococcus.

The invention also concerns a method for preventing or attenuating aninfection caused by a group B Streptococcus which comprisesadministering to an individual, suspected of being at risk for such aninfection, an effective amount of the conjugate vaccine of theinvention, such that it provides host immunity against the infection.

The invention further concerns a method for preventing or attenuatinginfection caused by a group B Streptococcus which comprisesadministering to a pregnant female an effective amount of a conjugatevaccine of the invention, such that it provides immunity to theinfection to an unborn offspring of the female.

The invention also provides a method for preventing or attenuating aninfection caused by a group B Streptococcus which comprisesadministering to an individual suspected of being at risk for such aninfection an effective amount of an antisera elicited from the exposureof a second individual to a conjugate vaccine of the invention, suchthat is provides host immunity to the infection.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 SEQ ID NOS. 30-47 shows the modifications of pUC12 to create theplasmid pUX12.

FIG. 2 shows the restriction and transcriptional map of the plasmidpUX12.

FIGS. 3A-3C SEQ ID NOS. 48-65 shows the modifications which were made topUX12 in order to produce the +1 reading frame plasmid pUX12+1 FIG. 3Aand which produce the -1 reading frame plasmid pUX12-1 FIG. 3C. FIG 3Bshows a construction which is additionally capable of resulting in a -1reading frame plasmid.

FIG. 4 shows the result of mouse protection studies employing rabbitantisera against S1 and S23. Protection was observed in mice inoculatedwith anti-S1 antisera (p<0.002) or with anti-S23 antisera (p<0.022). Dueto the sample size used, this difference in the observed statisticalsignificance between the S1 and S23 experiments is not significant. Inthe Figure, the mice surviving per total tested is reported as afraction above each bar.

FIG. 5 shows the sequencing strategy and restriction endonuclease map ofbca. The partial restriction endonuclease map encompasses the region ofpJMS23 from an Nde I site to a Sty I site located at nucleotide 3594 forwhich the nucleotide sequence of bca and flanking region was determined.The open reading frame is illustrated by an open box. Transposon Tn5seq1mutations (triangles) serve to prime nucleotide sequencing in bothdirections from each of the insertions. The regions of sequence obtainedfrom oligonucleotide primers (open arrows) and the nested deletions(closed arrows) are also shown. Restriction endonuclease cleavage sitesare abbreviated as follows: A, Alu I; B, Bsm I; F, Fok I; H, HincII; N,Nde I; S, Sty I. bp, base pairs.

FIG. 6A-6C shows the nucleotide SEQ ID NO: 14! and deduced amino acidsequences SEQ ID NO: 15! of bca and the flanking regions. The DNA strandis shown 5' to 3', and nucleotides are listed on the upper linebeginning 78 base pairs upstream from the open reading frame. Thededuced amino acid sequence for the open reading frame is below thenucleic acid sequence. The G+C content of 40% and the codon usage aresimilar to other streptococcal genes (Hollingshead, S. K. et al., J.Biol. Chem. 261:1677-1686 (1986)). Highlighted features include the -10(TATAAT) promoter consensus site, ribosomal binding site (RBS), signalsequence, repeat region 1, the C terminus, with the termination codon(TAA) at position 3161, and two regions of dyad symmetry that arepotential transcriptional terminators.

FIG. 7A-7B show homologies to the putative signal sequences andC-terminal membrane anchor of the C protein alpha antigen, respectively.FIG. 7A: the N terminus of the C protein alpha antigen on the top line(sequence 1) SEQ ID NO: 16! and is compared with the followingGram-positive signal sequences (accession codes are listed for each ofthe sequence numbers): sequence 2 SEQ ID NO: 17!, the C protein betaantigen (S 15330; STRBAGBA) and four M proteins of group AStreptococcus; sequence 3 SEQ ID NO: 18!, ennX (STRENNX); sequence 4 SEQID NO: 19!, emm24 (STREMM24); sequence 5 SEQ ID NO: 20!, M1 (S06767);sequence 6 SEQ ID NO: 21!, S01260. Lysine (K) and arginine (R) residuespreceding the underlined hydrophobic stretch are in boldface type, asare serine (S) and threonine (T) residues preceding the probable signalcleavage sites. The probable cleavage site for the alpha signal isfollowing the valine at position 41; however, alternative cleavage sitesexist at positions 53-56. FIG. 7B: The C terminus of the C protein alphaantigen is shown on the top line (sequence 1) SEQ ID NO: 22! andcompared with the following Gram-positive membrane anchor peptides:sequence 2 SEQ ID NO: 23!, M5 (A28616, M6 (A26297), and M24 (A28549);sequence 3 SEQ ID NO: 24!, ennX (STREENX); sequence 4 SEQ ID NO: 25!,S00128, STRPROTG, and A26314; sequence 5 SEQ ID NO: 26!, spg (A24496);sequence 6 SEQ ID NO: 27!, arp4 (S05568) and emm49 (STRM49NX, STRMM24);and sequence 7 SEQ ID NO: 28!, emm12 (STR12M), M5, M6, M24, emm12,emm49, and ennX are all M proteins; arp4 is a binding protein of group AStreptococcus. S00128, STRPROTG, spg, and A26314 are IgG bindingproteins of group G Streptococcus. Sequence 8 SEQ ID NO: 29! illustratesthe membrane anchor for the beta antigen, which lacks the PPFFXXAA SEQID NO: 1! motif. Highlighted areas include lysine residues (K) precedingthe LPXTGE SEQ ID NO: 2! motif (boxed), the hydrophobic region(underlined) with the PPFFXXAA SEQ ID NO: 1! consensus (boxed andunderlined), and the terminal amino acid aspartic acid (D) or asparagine(N).

FIG. 8 shows a comparison of the cloned and native gene products of bca.Surface proteins of the A909 strain of group B Streptococcus (type 1a/C)and C protein alpha antigen clone pJMS23-1 were analyzed by SDS/PAGE andWestern blotting and were probed with the alpha antigen-specificmonoclonal antibody 4G8. Arrowheads illustrate an example of thedifference between proteins. Molecular mass markers (in kDa) are shownon the right.

FIG. 9 shows a schematic of the open reading frame of bca. Summary ofthe structural features of the open reading frame of the C protein alphaantigen based on analysis of the amino acid sequence deduced from thenucleotide sequence of bca. The numbers above the boxes indicate thenucleotide position, and the numbers below are the amino acid residuesof the mature protein within the open reading frame.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Significance and Clinical Perspective

Maternal immunoprophylaxis with a vaccine to group B Streptococcus hasbeen proposed as a potential route for protecting against infection bothin the mother and in the young infant through the peripartum transfer ofantibodies (Baker, C. J. et al., New Eng. J. Med. (Editorial)314(26):1702-1704 (1986); Baker, C. J. et al., New Eng. J. Med. 319:1180(1988); Baker, C. J. et al., J. Infect. Dis. 7:458 (1985)). As is thecase with other encapsulated bacteria, susceptibility to infectioncorrelates with the absence of type-specific antibody (Kasper, D. L., etal., J. Clin. Invest. 72:260-269 (1983), Kasper, D. L., et al.,Antibiot. Chemother. 35:90-100 (1985)). The lack of opsonically activetype-specific anti-capsular antibodies to group B Streptococcus is arisk factor for the development of disease following colonization withgroup B Streptococcus (Kasper, D. L. et al., J. Infec. Dis. 153:407-415(1986)).

One approach has been to vaccinate with purified type-specific capsularpolysaccharides. Methods of producing such vaccines, and the use of suchvaccines to immunize against group B Streptococcus are disclosed byKasper, D. L. (U.S. Pat. No. 4,207,414 and U.S. Pat. No. RE31672, andU.S. Pat. Nos. 4,324,887, 4,356,263, 4,367,221, 4,367,222, and4,367,223), by Carlo, D. J. (U.S. Pat. No. 4,413,057, European PatentPublication 38,265), and by Yavordios, D. et al. (European PatentPublication 71,515), all of which references are incorporated herein byreference.

Although the polysaccharide capsule of group B Streptococcus is wellcharacterized and has been shown to play a role in both virulence andimmunity (Kasper, D. L. J. Infect. Dis. 153:407 (1986)), these capsularcomponents have been found to vary in their immunogenicity dependingboth on the specific capsular type and on factors in the host's immunesystem (Baker, C. J, et al., Rev. of Infec. Dis. 7:458467 (1985)). Arecently completed clinical trial evaluating a capsular polysaccharidevaccine of group B Streptococcus showed an overall response rate of 63%and indicated that such a vaccine was not optimally immunogenic (BakerC. J, et al., New Eng. J. Med. 319(18):1180-1185 (1988)).

Differences in immunogenicity have also been observed with the capsularpolysaccharides of other bacteria. For example, the vaccine against thetype C meningococcal capsule is highly active while the group Bmeningococcal polysaccharide vaccine is not immunogenic (Kasper, D. L.et al., J. Infec. Dis. 153:407415 (1986)). T-cell independent functionsof the host's immune system are often required for mounting an antibodyresponse to polysaccharide antigens. The lack of a T-cell independentresponse to polysaccharide antigens may be responsible for the lowlevels of antibody against group B Streptococcus present in motherswhose children subsequently develop an infection with group BStreptococcus. In addition, children prior to 18 or 24 months of agehave a poorly developed immune response to T-cell independent antigens.

Determinants of Virulence and Immunity in group B Streptococcus

There are five serotypes of group B Streptococcus that share a commongroup specific polysaccharide antigen. However, antibody of the groupantigen is not protective in animal models. Lancefield originallyclassified group B Streptococcus into four serotypes (Ia, Ib, II andIII) using precipitin techniques. The composition and structure of theunique type-specific capsular polysaccharides for each of the serotypeswas subsequently determined (Jennings, H. J, et al., Biochem.22:1258-1264 (1983), Kasper, D. L. et al., J. Infec. Dis. 153:407-415(1986), Wessels, M. R, et al., Trans. Assoc. Amer. Phys. 98:384-391(1985)). Wilkinson defined a fifth serotype, Ic, by the identificationof a protein antigen (originally called the Ibc protein) present on allstrains of serotype Ib and some strains with the type Ia capsule(Wilkinson, H. W, et al., J. Bacteriol. 97:629-634 (1969), Wilkinson, H.W, et al., Infec. and Immun. 4:596604 (1971)). This protein was laterfound to vary in prevalence between the different serotypes of group BStreptococcus but was absent in serotype Ia (Johnson, D. R, et al., J.Clin. Microbiol 19:506-510 (1984)).

The nomenclature has recently been changed to classify the serotypes ofgroup B Streptococcus solely by the capsular type-specificpolysaccharides, and a fifth capsular type has also been described (typeIV) (Pritchard, D. G, et al., Rev. Infec. Dis. 10(8):5367-5371 (1988)).Therefore, the typing of group B Streptococus strains is no longer basedon the antigenic Ibc protein, which is now called the C protein. Thetype Ic strain is reclassified as serotype Ia on the basis of itscapsular polysaccharide composition, with the additional informationthat it also carries the C protein.

Immunological, epidemiological and genetic data suggest that thetype-specific capsule plays an important role in immunity to group BStreptococcus infections. The composition and structure of thetype-specific capsular polysaccharides and their role in virulence andimmunity have been the subjects of intensive investigation (Ferrieri, P.et al., Infec. Immun. 27:1023-1032 (1980), Krause, R. M, et al., J. Exp.Med. 142:165-179 (1975), Levy, N. J, et al., J. Infec. Dis. 149:851-860(1984), Wagner, B, et al., J. Gen. Microbiol. 118:95-105 (1980),Wessels, M. R, et al., Trans. Assoc. Amer. Phys. 98:384-391 (1985)).

Controversy has existed regarding the structural arrangement of thetype-specific and group B streptococcal polysaccharides on the cellsurface, on the immunologically important determinants with in thetype-specific polysaccharide, and on the mechanisms of capsuledetermined virulence of group B Streptococcus (Kasper, D. L. et al., J.Infec. Dis. 153:407-415 (1986)). To study the role of the capsule invirulence, Rubens et al. used transposon mutagenesis to create anisogeneic strain of type III group B Streptococcus that isunencapsulated (Rubens, C. E, et al., Proc. Natl. Acad. Sci. USA84:7208-7212 (1987)). They demonstrated that the loss of capsuleexpression results in significant loss of virulence in a neonatal ratmodel. However, the virulence of clinical isolates with similar capsularcomposition varies widely. This suggests that other bacterial virulencefactors, in addition to capsule, play a role in the pathogenesis ofgroup B Streptococcus.

A number of proteins and other bacterial products have been described ingroup B Streptococcus whose roles in virulence and immunity have notbeen established, CAMP (Christine Atkins-Much Peterson) factor, pigment(probably carotenoid), R antigen, X antigen, anti-phagocytic factors andpoorly defined "pulmonary toxins" (Ferrieri, P, et al., J. Exp. Med.151:56-68 (1980); Ferrieri, P. et al., Rev. Inf. Dis. 10(2):1004-1071(1988); Hill, H. R. et al., Sexually Transmitted Diseases, McGraw-Hill,pp. 397-407). The C proteins are discussed below.

Isogeneic strains of group B Streptococcus lacking hemolysin show nodecrease in virulence in the neonatal rat model (Weiser, J. N, et al.,Infec. and Immun. 55:2314-2316 (1987)). Both hemolysin and neuraminidaseare not always present in clinical isolates associated with infection.The CAMP factor is an extracellular protein of group B Streptococcuswith a molecule weight of 23,500 daltons that in the presence ofstaphylococcal beta-toxin (a sphingomyelinase) leads to the lysis oferythrocyte membranes. The gene for the CAMP factor in group BStreptococcus was recently cloned and expressed in E. coli (Schneewind,O, et al., Infec. and Immun. 56:2174-2179 (1988)). The role, if any, ofthe CAMP factor, X and R antigens, and other factors listed above in thepathogenesis of group B Streptococcus is not disclosed in the prior art(Fehrenbach, F. J, et al., In: Bacterial Protein Toxins, Gustav FischerVerlag, Stuttgart (1988); Hill, H. R. et al., Sexually TransmittedDiseases, McGraw-Hill, NY, pp. 397-407 (1984)).

The C protein(s) are a group of a cell surface associated proteinantigens of group B Streptococcus that were originally extracted fromgroup B Streptococcus by Wilkinson et al. (Wilkinson, H. W, et al., J.Bacteriol. 97:629-634 (1969), Wilkinson, H. W, et al., Infec. and Immun.4:596-604 (1971)). They used hot hydrochloric acid (HCl) to extract thecell wall and trichloroacetic acid TCA) to precipitate protein antigens.Two antigenically distinct populations of C proteins have beendescribed: (1) A group of proteins that are sensitive to degradation bypepsin but not by trypsin, and called either TR (trypsin resistant) oralpha (α). (2) Another group of group B Streptococcus proteins that aresensitive to degradation by both pepsin and trypsin, and called TS(trypsin sensitive) or beta (β) (Bevanger, L, et al., Acta Path.Microbiol. Scand Sect. B. 87:51-54 (1989), Bevanger, L, et al., ActaPath. Microbiol. Scand. Sect. B. 89:205-209 (1981), Bevanger, L. et al.,Acta Path. Microbiol. Scand. Sect. B. 91:231-234 (1983), Bevanger, L. etal., Acta Path. Microbiol. Scand. Sect. B. 93:113-119 (1985), Bevanger,L, et al., Acta Path. Microbiol. Immunol. Scand. Sect. B. 93:121-124(1985), Johnson, D. R, et al., J. Clin. Microbiol. 19:506-510 (1984),Russell-Jones, G. J, et al., J. Exp. Med. 160:1476-1484 (1984)).

In 1975, lancefield et al. used mouse protection studies with antiseraraised in rabbits to define the C proteins functionally for theirability to confer protective immunity against group B Streptococcusstrains carrying similar protein antigens (Lancefield, R. C, et al., J.Exp. Med. 142:165-179 (1975)). Numerous investigators have obtainedcrude preparations of antigenic proteins from group B Streptococcus,that have been called C proteins, by chemical extraction from the cellwall using either HCl or detergents (Bevanger, L, et al., Acta Path.Microbiol. Scand. Sect. B. 89:205-209 (1981), Bevanger, L. et al., ActaPath. Microbiol. Scand. Sect. B. 93:113-119 (1985), Russell-Jones, G. J,et al., J. Exp. Med. 160:14761484 (1984), Valtonen, M. V, et al.,Microb. Path. 1:191-204 (1986), Wilkinson, H. W, et al., Infec. andImmun. 4:596-604 (1971)). The reported sizes for these antigens havevaried between 10 and 190 kilodaltons, and a single protein species hasnot been isolated or characterized (Ferrieri, P. et al., Rev. Inf. Dis.10(2):1004-1071 (1988)).

By screening with protective antisera, C proteins can be detected inabout 60% of clinical isolates of group B Streptococcus, and are foundin all serotypes but with differing frequencies (Johnson, D. R, etal.,J. Clin. MicrobioL 19:506-510 (1984)). Individual group B Streptococcusisolates may have both the TR and TS antigens, or only one, or neitherof these antigens. Except for the ability of the partially purifiedantigens to elicit protective immunity, the role of these antigens inpathogenesis has not been studied in vitro. In Wvo studies with group BStreptococcus strains that carry C proteins provides some evidence thatthe C proteins may be responsible for resistance to opsonization (Payne,N. R, et al., J. Infec. Dis. 151:672-681 (1985)), and the C proteins mayinhibit the intracellular killing of group B Streptococcus followingphagocytosis (Payne, N. R, et al., Infect. and Immun. 55:1243-1251(1987)). It has been shown that type II strains of group B Streptococcuscarrying the C proteins are more virulent in the neonatal rat sepsismodel (Ferrieri, P, et al., Infect. Immun. 27:1023-1032 (1980),Ferrieri, P. et al., Rev. Inf. Dis. 10(2):1004-1071 (1988)). Since thereis no genetic data on the C proteins, isogeneic strains lacking the Cproteins have not previously been studied. There is evidence that one ofthe TS, or β, C proteins binds to IgA (Russell-Jones, G. J, et al., J.Exp. Med. 160:1476-1484 (1984)). The role, if any, that the binding ofIgA by the C proteins has on virulence is, however, not disclosed.

In 1986, Valtonen et al. isolated group B Streptococcus proteins fromculture supernatants that elicit protection in the mouse model(Valtonen, M. V, et al., Microb. Path. 1:191-204 (1986)). Theyidentified, and partially purified, a trypsin resistant group BStreptococcus protein with a molecular weight of 14,000 daltons.Antisera raised to this protein in rabbits protected mice againstsubsequent challenge with type Ib group B Streptococcus (89%protection). This protein is, by Lancefield's definition, a C protein.However, when antisera raised against this protein were used toimmunoprecipitate extracts of group B Streptococcus antigens, a numberof higher molecular weight proteins were found to be reactive. Thissuggested that the 14,000 m.w. protein may represent a common epitope ofseveral group B Streptococcus proteins, or that it is a degradationproduct found in the supernatants of group B Streptococcus cultures. Thediversity in the sizes in C proteins isolated from both the bacterialcells and supernatants suggests that the C proteins may represent a genefamily, and maintain antigenic diversity as a mechanism for protectionagainst the immune system.

The range of reported molecular weights and difficulties encountered inpurifying individual C proteins are similar to the problems that manyinvestigators have faced in isolating the M protein of group AStreptococcus (Dale, J. B, et al., Infec. and Inmun. 46(1):267-269(1984), Fischetti, V. A, et al., J. Exp. Med. 144:32-53 (1976),Fischetti, V. A, et al., J. Exp. Med 146:1108-1123 (1977)). The gene forthe M protein has now been cloned and sequenced, and found to contain anumber of repeated DNA sequences (Hollingshead, S. K, et al., J. Biol.Chem. 261:1677-1686 (1986), Scott, J. R, et al., Proc. Natl. Acad. SciUSA 82:1822-1826 (1986), Scott, J. R, et al., Infec. and Immunn.52:609-612 (1986)). These repeated sequences may be responsible forpost-transcriptional processing that results in a diversity in the sizeof M proteins that are produced. The mechanism by which this occurs isnot understood. The range of molecular weights described for the Cproteins of group B Streptococcus might result from a similar process.

Cleat et al. attempted to clone the C proteins by using two preparationsof antisera to group B Streptococcus obtained from Bevanger (α and β) toscreen a library of group B Streptococcus DNA in E. coli (Bevanger, L.et al., Acta Path. Microbiol. Immunol. Scand. Sect. B. 93:113-119(1985), Cleat, P. H, et al., Infec. and Innun. 55(5): 1151-1155 (1987),which references are incorporated herein by reference). Theseinvestigators described two clones that produce proteins that bind toantistreptococcal antibodies. However, they failed to determine whethereither of the cloned proteins had the ability to elicit protectiveantibody, or whether the prevalence of these genes correlated the withgroup B Streptococcus strains known to carry the C proteins. The role ofthe cloned gene sequences in the virulence of group B Streptococcus wasnot investigated. Since the C proteins are defined by their ability toelicit protective antibodies, this work failed to provide evidence thateither of the clones encodes a C protein.

The Conjugated Vaccine of the Present Invention

The present invention surmounts the above-discussed deficiencies ofprior vaccines to group B Streptococcus through the development of aconjugate vaccine in which the capsular polysaccharides are covalentlylinked to a protein backbone. This approach supports the development ofa T-cell dependent antibody response to the capsular polysaccharideantigens and circumvents the T-cell independent requirements forantibody production (Baker, C. J, et al., Rev. of Infec. Dis. 7:458-467(1985), Kasper, D. L. et al., J. Infec. Dis. 153:407415 (1986), whichreferences are incorporated herein by reference).

In a conjugate vaccine, an antigenic molecule, such as the capsularpolysaccharides of group B Streptococcus (discussed above), iscovalently linked to a "carrier" protein or polypeptide. The linkageserves to increase the antigenicity of the conjugated molecule. Methodsfor forming conjugate vaccines from an antigenic molecule and a"carrier" protein or polypeptide are known in the art (Jacob, C. O, etal., Eur. J. Immunol. 16:1057-1062 (1986); Parker J. M. R. et al., In:Modem Approaches to Vaccines, Chanock, R. M. et al., eds, pp. 133-138,Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. (1983);Zurawski, V. R, et al., J. Immunol. 121:122-129 (1978); Klipstein, F. A,et al., Infect. Immun. 37:550-557 (1982); Bessler, W. G, Immunobiol.170:239-244 (1985); Posnett, D. N, et al., J. Biol. Chem. 263:1719-1725(1988); Ghose, A. C, et al., Molec. Immunol. 25:223-230 (1988); all ofwhich references are incorporated herein by reference).

A prototype model for conjugate vaccines was developed againstHemophilus influenzae (Anderson, P, Infec. and Immun. 39:223-238 (1983);Chu, C, et al., Infect. Immun. 40:245-256 (1983); Lepow, M, Pediat.Infect. Dis. J. 6:804-807 (1987), which references are incorporatedherein by reference), and this model may be employed in constructing thenovel vaccines of the present invention. Additional methods forproducing such a conjugate vaccine are disclosed by Anderson, P. W, etal., European Patent Publication 245,045; Anderson, P. W, et al., U.S.Pat. Nos. 4,673,574 and 4,761,283; Frank, R. et al., U.S. Pat. No.4,789,735; European Patent Publication No. 206,852; Gordon, L. K, U.S.Pat. No. 4,619,828; and Beachey, E. H, U.S. Pat. No. 4,284,537, all ofwhich references are incorporated herein by reference.

The protein backbones for conjugate vaccines such as the Hemophilusinfluenzae vaccine have utilized proteins that do not share antigenicproperties with the target organism from which the bacterial capsularpolysaccharides were obtained (Ward, J. et al., In: Vaccines, Plotkin,S. A, et al., eds, Saunders, Philadelphia, page 300 (1988).

In contrast, the conjugate vaccine of the present invention employsimmunogenic proteins of group B Streptococcus as the backbone for aconjugate vaccine. Such an approach is believed to lead to moreeffective vaccines (Insel, R. A, et al., New Eng. J. Med. (Editorial)319(18):1219-1220 (1988)). The conjugate, protein-polysaccharide vaccineof the present invention is the first to specifically characterize groupB Streptococcus proteins that may be used in a conjugate vaccine. Anyprotein which is characteristic of group B Streptococcus may be employedas the protein in the conjugate vaccines of the present invention. Itis, however, preferred to employ a C protein of a group B Streptococcusfor this purpose. As discussed more fully below, plasmids pJMS1 andpJMS23 contain DNA which encode Streptococcus C protein. The mostpreferred C proteins are those obtained upon the expression of such DNAin bacteria.

As indicated above, the present invention concerns the cloning andexpression of genes which encode the protective group B Streptococcusprotein antigens. Such proteins are preferably used as the proteinbackbone to which the one or more of the polysaccharides of the group BStreptococcus can be conjugated in order to form a conjugate vaccineagainst these bacteria. Alternatively, one or more proteins as describedherein may be conjugated to the structure of a polysaccharide of thegroup B Streptococcus.

The role of these proteins in the virulence and immunity of group BStreptococcus may be exploited to develop an additional therapy againstgroup B Streptococcus infection. The isolation and characterization ofthese genes of a bacterial origin allows the manipulation of the geneproducts to optimize both the adjuvant and antigenic properties of thepolypeptide backbone/carrier of the conjugate vaccine.

Genetic Studies of the C Proteins

The present invention thus concerns the cloning of the C proteins ofgroup B Streptococcus, their role in virulence and immunity, and theirability to serve as an immunogen for a conjugate vaccine against group BStreptococcus.

Despite the extensive literature available on cloning in many groups ofStreptococci, only limited genetic manipulations have been accomplishedin group B Streptococcus (Macrina, F. L, Ann. Rev. Microbiol. 38:193-219(1984), Wanger, A. R, et al., Infec. and Immun. 55:1170-1175 (1987)).The most widely used technique in group B Streptococcus has been thedevelopment of Tn916 and its use in transposon mutagenesis (Rubens, C.E, et al., Proc. Natl. Acad. Sci. USA 84:7208-7212 (1987), Wanger, A. R,et al., Res. Vet. Sci. 38:202-208 (1985)). However, since it wouldappear that there is more than one gene for the C proteins and theprotective antisera bind to several proteins, screening for the Cprotein genes by transposon mutagenesis is impractical.

The present invention accomplishes the cloning of the C proteins (and ofany other proteins which are involved in the virulence of the group BStreptococcus, or which affect host immunity to the group BStreptococcus) through the use of a novel plasmid vector. For thispurpose, it is desirable to employ a cloning vector that could berapidly screened for expression of proteins which bind to naturallyelicited antibodies to group B Streptococcus. Since such antibodies areheterologous polyclonal antibodies and not monoclonal antibodies, it wasnecessary that a vector be employed which could be easily screenedthrough many positive clones to identify genes of interest.

A number of techniques were available for screening clones for theexpression of antigens that bind to a specific antisera (Aruffo, A, etal., Proc. Natl. Acad. Sci. USA 84:8573-8577 (1987)). The most widelyused system, λgt11, was developed by Young and Davis (Huynh, T. V. etal., In: DNA Cloning, A Practical Approach, Vol. 1 (Glover, D. M, Ed.)IRL Press, Washington pp. 49-78 (1985); Wong, W. W, et al., J. Immunol.Methods. 82:303-313 (1985), which references are incorporated herein byreference). This technique allows for the rapid screening of clonesexpressed in the lysogenic phage whose products are released by phagelysis. Commonly faced problems with this system include the requirementfor subdloning DNA fragments into plasmid vectors for detailedendonuclease restriction mapping, preparing probes and DNA sequencing.In addition, the preparation of DNA from phage stocks is cumbersome andlimits the number of potentially positive clones that can be studiedefficiently. Finally, the preparation of crude protein extracts fromcloned genes is problematic in phage vector hosts.

To circumvent these problems, the present invention provides a plasmidvector which was developed for screening cloned bacterial chromosomalDNA for the expression of proteins involved in virulence and/orimmunity. The present invention thus further concerns the developmentand use of an efficient cloning vector that can be rapidly screened forexpression of proteins which bind to naturally elicited antibodies togroup B Streptococcus. The vector was prepared by modifying the commonlyused plasmid cloning vector, pUC12 (Messing, J, et al., Gene 19:269-276(1982); Norrander, J, et al., Gene 26: 101-106 (1983); Vieira, J, etal., Gene 19:259-268 (1982); which references are incorporated herein byreference). The invention concerns the vector described below, and itsfunctional equivalents.

Using this system, plasmid clones can be easily manipulated, mapped withrestriction endonucleases and their DNA inserts sequences, probesprepared and gene products studied without the necessity for subcloning.pUC12 is a 2.73 kilobase (kb) high copy number plasmid that carries aColE1 origin of replication, ampicillin resistance and a polylinker inthe lacZ gene (Ausubel, F. M, et al., Current Topics in MolecularBiology; Greene Publ. Assn./ Wiley Interscience, NY (1987) whichreference is incorporated herein by reference).

Several modifications were made in the polylinker of pUC12 (Aruffo, A,et al., Proc. Natl. Acad. Sci. USA 84:8573-8577 (1987) which referenceis incorporated herein by reference). The overall plan in altering pUC12was to modify the polylinker to present identical but non-cohesive BstXIsites for cloning, to add a "stuffer" fragment to allow for easyseparation of the linear host plasmid, and to provide for expressionfrom the lac promoter in all three translational reading frames.

In order to provide a site for the insertion of foreign DNA with a highefficiency and to minimize the possibility for self-ligation of theplasmid, inverted, non-cohesive BstXI ends were added to the polylinker.As shown in FIG. 1, pUC12 was first cut with BamHI (Step 1) and theplasmid was mixed with two synthetic oligonucleotide adaptors that arepartially complementary: a 15-mer (GATCCATTGTGCTGG) SEQ ID NO: 3! and an11-mer (GTAACACGACC) SEQ ID NO: 4! (Step 2). When the adaptors areligated into pUC12, two new BstI sites are created but the originalBamHI sites are also restored (Step 3). The plasmid was then treatedwith polynucleotide kinase and ligated to form a closed circular plasmid(Step 4). When this plasmid is treated with BstXI, the resulting endsare identical and not cohesive (both have GTGT overhangs) (Step 5).

A second modification in the polylinker was done to allow for thepurification of the linear plasmid for cloning without contaminationfrom partially cut plasmid that can self-ligate. A blunt end, 365 basepair (bp), FnuD2 fragment was obtained from the plasmid pCDM. Thiscassette or "stuffer" fragment, which does not contain a BstXI site, wasblunt end ligated to two synthetic oligonucleotides that are partiallycomplementary: a 12-mer (ACACGAGATTTC) SEQ ID NO: 5! and an 8-mer(CTCTAAAG) (Step 6). The resulting fragment with adaptors has 4 bpoverhangs (ACAC) that are complementary to the ends of the modifiedpUC12 plasmid shown in Step 5. The modified pUC12 plasmid was ligated tothe pCDM insert with adaptors; the resulting construct, named pUX12, isshown in FIG. 2. The pUX12 plasmid can be recreated from plasmids pJMS1or pJMS23 by excision of the introduced Streptococcus DNA sequences.Alternatively, it may be formed by recombinant methods (or by homologousrecombination), using plasmid pUC12.

Since pUX12 is to be used as an expression vector, it is preferable tofurther modified the polylinker such that it will contain all threepotential reading frames for the lac promoter. These changes allow forthe correct translational reading frame for cloned gene fragments with afrequency of one in six. For example, a cloned fragment can insert inthe vector in one of two orientations and one of three reading frames.To construct a +1 reading frame, the pUX12 plasmid was cut with therestriction enzyme EcoRI which cleaves at a unique site in thepolylinker. The single stranded 5' sticky ends were filled in using the5'-3' polymerase activity of T4 DNA polymerase, and the two blunt endsligated. This resulted in the loss of the EcoRI site, and the creationof a new XmnI site (FIG. 3A). This construction was confirmed bydemonstrating the loss of the EcoRI site and confirming the presence ofa new XmnI site in the polylinker. In addition, double stranded DNAsequencing on the +1 modified pUX12 plasmid was performed using standardsequencing primers (Ausubel, F. M, et al., Current Topics in MolecularBiology; Greene Publ. Assn./ Wiley Interscience, NY (1987)). The DNAsequence showed the addition of 4 base pairs to the polylinker andconfirmed the modification of pUX12 to a +1 reading frame. This plasmidis called pUX12+1.

In order to construct a -1 reading frame, the pUX12 vector was cut withthe restriction enzyme SacI which cuts at a unique site in thepolylinker of pUX12. The single stranded 3' sticky ends were cut back toblunt ends using the 3'-5' exonuclease activity of T4 polymerase, andthe resulting blunt ends ligated. The resulting sequence shouldeliminate the SacI site while resulting in a new FnuD2 site (FIG. 3B).However, restriction mapping of the pUX12-1 plasmids showed that whilethe SacI site was absent, there was no FnuD2 site present. In addition,the SmaI/XmaI sites on the polylinker were no longer present. Severalpotential pUX12-1 constructs were sequenced from mini-prep,double-stranded DNA. Of the six modified plasmids sequenced, one wasfound with ten nucleotides absent, thereby creating a -1 reading frame(FIG. 3C). This suggests that the T4 DNA polymerase has additionalexonuclease activity and cuts back additional double stranded portionsof the polylinker. Nevertheless, the resulting plasmid had a -1 readingframe. The plasmid was named pUX12-1.

The use of the pUX12 vectors in the cloning of antigenic proteins ofgroup B Streptococcus are discussed in detail in the Examples below. Inbrief, DNA derived from group B Streptococcus, or complementary to suchDNA is introduced into the pUX12, pUX12+1 or pUX12-1 vectors andtransformed into Eschelichia coli. The cloned DNA is expressed in E.coli and the cellular lysate is tested to determine whether it containsany protein capable of binding to antisera to group B Streptococcits.

There are a number of potentially interesting modifications of pUX12that could increase its utility. For example, the lac promoter could bereplaced by another promoter, the origin of replication could bemodified to produce a lower copy number vector and the drug resistancemarker could be changed.

Any vector capable of providing the desired genetic information to thedesired host cell may be used to provide genetic sequences encoding thealpha antigen derivatives of the invention to a host cell. For example,in addition to plasmids, such vectors include linear DNA, cosmids,transposons, and phage.

The host cell is not limited to E. coli. Any bacterial or yeast (such asS. cerewsiae) host that is capable of expressing the derivatives of theinvention may be used as an appropriate host. For example, B. subtilisand the group B Streptococcus may be used as hosts. Methods for cloningand into such hosts are known. For example, for Gram-positive hosts, seeHarwood, C. R., et al., eds., "Molecular Biological Methods forBacillus," Wiley-Interscience, New York, 1991) for a description ofculture methods, genetic analysis plasmids, gene cloning techniques, theuse of transposons, phage, and integrational vectors for mutagenesis andthe construction of gene fusions, and methods of measuring geneexpression. Appropriate hosts are available from stock centers such asthe American Type Culture Collection (Rockville, Md., USA) and theBacillus Genetic Stock Center (Ohio State Univ., Columbus, Ohio, USA).

The present invention concerns a vaccine comprising a polysaccharide(such as the capsular polysaccharide) which is characteristic of thegroup B Streptococcus conjugated to a protein which is alsocharacteristic of the group B Streptococcus. The "polysaccharide" and"protein" of such a conjugated vaccine mnay be identical to a moleculewhich is characteristic of the group B Streptococcus, or they may befunctional derivatives of such molecules.

For the purposes of the present invention, a group B Streptococcuspolysaccharide is any group B-specific or type-specific polysaccharide.Preferably, such polysaccharide is one which, when introduced into amammal (either animal or human) elicits antibodies which are capable ofreacting with group B Streptococcus may be employed. Examples of thepreferred polysaccharides of the present invention include the capsularpolysaccharide of the group B Streptococcus, or their equivalents. Forthe purposes of the present invention, any protein which when introducedinto a mammal (either animal or human) either elicits antibodies whichare capable of reacting a protein expressed by group B Streptococcus, orwhich increases the immunogenicity of a polysaccharide to elicitantibodies to a polysaccharide of the group B Streptococcus may beemployed. Examples of the preferred proteins of the present inventioninclude the C proteins of the group B Streptococcus, or theirequivalents.

Examples of functional derivatives of the peptide antigens includefragments of a natural protein, such as N-terminal fragment, C-terminalfragment or internal sequence fragments of the group B Streptococcus Cprotein alpha antigen that retain their ability to elicit protectiveantibodies against the group B Streptococcus. The term functionalderivatives is also intended to include variants of a natural protein(such as proteins having changes in amino acid sequence but which retainthe ability to elicit an immunogenic, virulence or antigenic property asexhibited by the natural molecule), for example, the variants of thealpha antigen recited below that possess fewer of the internal repeatsthan does the native alpha antigen, and/or an altered flanking sequence.

The peptide antigen that is conjugated to the polysaccharide in thevaccine of the invention may be a peptide encoding the native amino acidsequence of the alpha antigen, as encoded on plasmid pJMS23 (with orwithout the signal peptide sequence) or it may be a functionalderivative of the native sequence. The native group B Streptococcus Cprotein alpha antigen as encoded on pJMS23 contains an open readingframe of 3060 nucleotides and encodes a precursor protein of 108,705daltons. Cleavage of the putative signal sequence of 41 amino acidsyields a mature protein of 104,106 daltons. The 20,417 dalton N-terminalregion of the alpha antigen shows no homology to previously describedprotein sequences and is followed by a series of nine tandem repeatingunits that make up 74% of the mature protein. Each repeating unit(denoted herein as "R") is identical and consists of 82 amino acids witha molecular mass of 8665 daltons, which is encoded by 246 nucleotides.The size of the repeating units corresponds to the observed sizedifferences in the heterogeneous ladder of alpha C proteins naturallyexpressed by the group B Streptococcus. The C-terminal region of thealpha antigen contains a membrane anchor domain motif that is shared bya number of Gram-positive surface proteins. The large region ofidentical repeating units in this gene, (termed the bca gene, for groupB Streptococcus, C protein, alpha antigen) defines protective epitopesand may be used to generate diversity of alpha antigen functionalderivatives that are useful in the vaccines of the invention.

Preferably, the sequence of such a functional alpha antigen derivativecontains 1-9 copies of the 82 amino acid repeat (246 nucleotides) thatbegin at amino acid 227 of the DNA sequence of FIG. 6A-6C, (as usedherein, the partial repeat designed as repeat 9' therein is also usefulin this regard). The functional derivative may lack the 185 amino acid5' flanking sequence (555 nucleotides) that is found in the nativeprotein prior to the repeating sequence or it may retain this sequenceand/or the derivative may lack the 45 amino acid (246 nucleotides)C-terminal anchor sequence or it may retain this sequence. Thefunctional derivative may be the N-terminal fragment that precedes thestart of the alpha antigen repeating unit(s) or the functionalderivative may be only the C-terminal fragment that follows the end ofthe alpha antigen repeating unit(s) or the function derivative may be ahybrid of the N-terminal fragment and C-terminal fragment with no copiesof the "R" units as defined below. The amino terminal sequence of thenative alpha antigen may or may not contain the signal sequence. Eitherof the alpha antigen's amino terminal sequence or carboxy terminalsequence may be used in the conjugate vaccines of the invention, with orwithout one or more copies of the sequence that is repeated in the coreof the native alpha antigen protein.

As used herein, "R" represents one copy of the 82 amino acid repeat thatbeings at amino acid 227 of the alpha antigen DNA sequence of FIG.6A-6C, "R_(x) " represents "X" number of tandem copies of this repeat,tandemly joined at the carboxyl end of one R unit to the amino terminalend of the adjoining R unit, "N" represents the 5' amino acid flankingsequence that is found in the sequence shown on FIG. 6A-6C, with orwithout the signal sequence; when the signal sequence is lacking, "N" isa 185 amino acid 5' flanking sequence that is found in the nativeprotein as shown on FIG. 6A-6C; when the signal sequence is present, "N"is a 226 amino acid 5' flanking sequence as shown in FIG. 6A-6C. "C"represents the 45 amino acid C-terminal anchor sequence as shown on FIG.6. Using this notation, the following species are examples ofderivatives of the native protein that may be constructed according tothe invention:

1. R₁

2. N

3. C

4. N--C

5. N--R₁

6. R--C

7. N--R₁ --C

8. R₂

9. N--R₂

10. R₂ --C

11. N--R₂ --C

12. R₃

13. N--R₃

14. R₃ --C

15. N--R₃ --C

16. R₄

17. N--R₄

18. R₄ --C

19. N--R₄ --C

20. R₅

21. N--R₅

22. R₅ --C

23. N--R₅ --C

24. R₆

25. N--R₆

26. R₆ --C

27. N--R₆ --C

28. R₇

29. N--R₇

30. R₇ --C

31. N--R₇ --C

32. R₈

33. N--R₈

34. R₈ --C

35. N--R₈ --C

36. R₉

37. N--R₉

38. R₉ --C

39. N--R₉ --C

40. R₁₀

41. N--R₁₀

42. R₁₀ --C

43. N--R₁₀ --C.

Greater than 10 repeating R units, including, for example, 11, 12, 13,14, 15, 16, 17, 18, 19 or 20 R units, may be constructed in a similarmanner. In addition, fragments of R, N, or C may be used if suchfragments enhance the functional ability of the derivative to elicitprotective antibodies against the group B Streptococcus, or if suchfragment provides another desired property to the construct, such as asecretion signal or membrane localization signal.

Alpha antigens from other strains of the group B Streptococcus may beprepared and used in a similar manner as a slight variability in thesequence of the protein, such as in the N terminus or C terminus or Rrepeat would not alter the biological properties and their functionalability to elicit protective antibodies. For example, a group BStreptococcus alpha antigen isolated from a different strain of thegroup B Streptococcus and having the same repeat unit but a differentN-terminal amino acid sequence is intended to be within the scope of theinvention.

The peptides of the invention, whether encoding a native protein or afunctional derivative thereof, are conjugated to a group B Streptococcuscarbohydrate moiety by any means that retains the ability of theseproteins to induce protective antibodies against the group BStreptococcus.

Heterogeneity in the vaccine may be provided by mixing specificconjugated species. For example, the vaccine preparation may contain oneor more copies of one of the peptide forms conjugated to thecarbohydrate, or the vaccine preparation may be prepared to contain morethan one form of the above functional derivatives and/or the nativesequence, each conjugated to a polysaccharide used therein. Conjugatesproviding a peptide (such as one of the peptides exemplified in groupnumbers 1-43) can be mixed with conjugates providing any other peptide(such as a second example from group numbers 1-43) to arrive at a"compound" conjugate vaccine. A multivalent vaccine may also be preparedby mixing the group B-specific conjugates as prepared above with otherproteins, such as diphtheria toxin or tetanus toxin, and/or otherpolysaccharides, using techniques known in the art.

Heterogeneity in the vaccine may also be provided by utilizing group BStreptococcal preparations from group B Streptococcal hosts (especiallyinto Streptococcus agalactine), that have been transformed with therecombinant constructs of the invention such that the streptococcal hostexpresses the alpha antigen protein or functional derivative thereof. Insuch cases, homologous recombination between the genetic sequencesencoding the repeating R units will result in spontaneous mutation ofthe host, such that a population of hosts is easily generated and suchhosts express a wide range of antigenic alpha antigen functionalderivatives useful in the vaccines of the invention. Such spontaneousmutation usually results in the deletion of R units, or portionsthereof, although mutation of other regions of the alpha antigen mayalso occur.

As used herein, a polysaccharide or protein is "characteristic" of abacteria if it is substantially similar in structure or sequence to amolecule naturally associated with the bacteria. The term is intended toinclude both molecules which are specific to the organism, as well asmolecules which, though present on other organisms, are involved in thevirulence or antigenicity of the bacteria in a human or animal host.

The vaccine of the present invention may confer resistance to group BStreptococcus by either passive immunization or active immunization. Inone embodiment of passive immunization, the vaccine is provided to ahost (i.e. a human or mammal) volunteer, and the elicited antisera isrecovered and directly provided to a recipient suspected of having aninfection caused by a group B Streptococcus.

The ability to label antibodies, or fragments of antibodies, with toxinlabels provides an additional method for treating group B Streptococcusinfections when this type of passive immunization is conducted. In thisembodiment, antibodies, or fragments of antibodies which are capable ofrecognizing the group B Streptococcus antigens are labeled with toxinmolecules prior to their administration to the patient. When such atoxin derivatized molecule binds to a group B Streptococcus cell, thetoxin moiety will cause the death of the cell.

In a second embodiment, the vaccine is provided to a female (at or priorto pregnancy or parturition), under conditions of time and amountsufficient to cause the production of antisera which serve to protectboth the female and the fetus or newborn (via passive incorporation ofthe antibodies across the placenta).

The present invention thus concerns and provides a means for preventingor attenuating infection by group B Streptococcus, or by organisms whichhave antigens that can be recognized and bound by antisera to thepolysaccharide and/or protein of the conjugated vaccine. As used herein,a vaccine is said to prevent or attenuate a disease if itsadministration to an individual results either in the total or partialattenuation (i.e. suppression) of a symptom or condition of the disease,or in the total or partial immunity of the individual to the disease.

The administration of the vaccine (or the antisera which it elicits) maybe for either a "prophylactic" or "therapeutic" purpose. When providedprophylactically, the compound(s) are provided in advance of any symptomof group B Streptococcus infection. The prophylactic administration ofthe compound(s) serves to prevent or attenuate any subsequent infection.When provided therapeutically, the compound(s) is provided upon thedetection of a symptom of actual infection. The therapeuticadministration of the compound(s) serves to attenuate any actualinfection.

The protective-antibody-eliciting agents of the present invention may,thus, be provided either prior to the onset of infection (so as toprevent or attenuate an anticipated infection) or after the initiationof an actual infection.

A composition is said to be "pharmacologically acceptable" if itsadministration can be tolerated by a recipient patient. Such an agent issaid to be administered in a "therapeutically effective amount" if theamount administered is physiologically significant. An agent isphysiologically significant if its presence results in a detectablechange in the physiology of a recipient patient.

As would be understood by one of ordinary skill in the art, when thevaccine of the present invention is provided to an individual, it may bein a composition which may contain salts, buffers, adjuvants, or othersubstances which are desirable for improving the efficacy of thecomposition. Adjuvants are substances that can be used to specificallyaugment a specific immune response. Normally, the adjuvant and thecomposition are mixed prior to presentation to the immune system, orpresented separately, but into the same site of the animal beingimmunized. Adjuvants can be loosely divided into several groups basedupon their composition. These groups include oil adjuvants (for example,Freund's complete and incomplete), mineral salts (for example,AlK(SO₄)₂, AlNa(SO₄)₂, AlNH₄ (SO₄), silica, kaolin, and carbon),polynucleotides (for example, poly IC and poly AU acids), and certainnatural substances (for example, wax D from Mycobacterium tuberculosis,as well as substances found in Corynebactenium parvum, or Bordetellapertussis, and members of the genus Brucella. Among those substancesparticularly useful as adjuvants are the saponins such as, for example,Quil A. (Superfos A/S, Denmark). Examples of materials suitable for usein vaccine compositions are provided in Remington's PharmaceuticalSciences (Osol, A, Ed, Mack Publishing Co, Easton, Pa., pp. 1324-1341(1980), which reference is incorporated herein by reference).

The therapeutic compositions of the present invention can beadministered parenterally by injection, rapid infusion, nasopharyngealabsorption (intranasopharangeally), dermoabsorption, or orally. Thecompositions may alternatively be administered intramuscularly, orintravenously. Compositions for parenteral administration includesterile aqueous or non-aqueous solutions, suspensions, and emulsions.Examples of non-aqueous solvents are propylene glycol, polyethyleneglycol, vegetable oils such as olive oil, and injectable organic esterssuch as ethyl oleate. Carriers or occlusive dressings can be used toincrease skin permeability and enhance antigen absorption. Liquid dosageforms for oral administration may generally comprise a liposome solutioncontaining the liquid dosage form. Suitable forms for suspendingliposomes include emulsions, suspensions, solutions, syrups, and elixirscontaining inert diluents commonly used in the art, such as purifiedwater. Besides the inert diluents, such compositions can also includeadjuvants, wetting agents, emulsifying and suspending agents, orsweetening, flavoring, or perfuming agents.

Many different techniques exist for the timing of the immunizations whena multiple administration regimen is utilized. It is possible to use thecompositions of the invention more than once to increase the levels anddiversities of expression of the immunoglobulin repertoire expressed bythe immunized animal. Typically, if multiple immunizations are given,they will be given one to two months apart.

According to the present invention, an "effective amount" of atherapeutic composition is one which is sufficient to achieve a desiredbiological effect. Generally, the dosage needed to provide an effectiveamount of the composition will vary depending upon such factors as theanimal's or human's age, condition, sex, and extent of disease, if any,and other variables which can be adjusted by one of ordinary skill inthe art.

The antigenic preparations of the invention can be administered byeither single or multiple dosages of an effective amount. Effectiveamounts of the compositions of the invention can vary from 0.01-1,000μg/ml per dose, more preferably 0.1-500 μg/ml per dose, and mostpreferably 10-300 μg/ml per dose.

Having now generally described the invention, the same will be morereadily understood through reference to the following examples which areprovided by way of illustration, and are not intended to be limiting ofthe present invention, unless specified.

EXAMPLE 1 Cloning Efficiency of the pUX12 Vectors

Several experiments were designed to test the cloning efficiency of thepUX12 vectors and to determine whether the modified reading framestranscribed correctly. The results of these experiments will be brieflysummarized below:

1. To clone a DNA fragment into pUX12, the three constructs, pUX12 (theoriginal "zero" reading frame construction), pUX12+1 and pUX12-1, weremixed in equimolar concentrations. The plasmids were then cut with BstXIto cleave the stuffer fragment within the polylinker. The stufferfragment was separated from the plasmid using either low melting pointagarose or a potassium acetate gradient (Aruffo, A, et al., Proc. Natl.Acad. Sci. USA 84:8573-8577 (1987), Ausubel, F. M, et al., CurrentTopics in Molecular Biology; Greene Publ. Assn./ Wiley Interscience, NY(1987)). The DNA to be cloned was cut with a restriction enzyme thatgives blunt ends (any such restriction enzyme may be employed). Ifnecessary, double stranded DNA with signal stranded ends can be modifiedto create blunt ends. The blunt ends of the DNA fragments were mixedwith the two synthetic oligonucleotide adaptors. These are the same12-mer and 8-mer used in preparing the stuffer fragment. The modifiedDNA fragments were separated from the unincorporated syntheticoligonucleotides on a potassium acetate gradient. These fragments werethen ligated into the linear pUX12 family of plasmids and used totransform E. coli.

To verify that the pUX12 vectors self-ligate at a low frequency underconditions optimize for the cloning of inserts with adaptors, a seconddrug resistance marker was cloned into pUX12. As shown in FIG. 1, pUX12has a β-lactamase gene and carriers resistance to ampicillin (amp^(R)).The rationale for cloning a second marker was to compare the ratio ofclones that contained both drug resistance markers to those pUX12plasmids that self-ligated under typical cloning conditions andtherefore only expressed resistance to ampicillin. The tetracyclineresistance gene (tet^(R)) from the plasmid pBR322 was cloned into thepolylinker of pUX12 with the adaptors described above. A group of testligations were run to establish the optimal concentration ofoligonucleotide adaptor to fragment ends, and the ratio of modifiedinsert to linear pUX12 plasmid for ligation and transformation. By usingthe tet^(R) gene as a marker, we were able to determine cloningparameters so that greater than 99% of the transformants selected onampicillin containing plates also carried the tet^(R) marker. Thus, thefrequency of self-ligation is very low in this system and it is notnecessary to screen for the presence of an insert in the polylinkerprior to screening a library in pUX12.

2. To confirm the position of the translational reading frame in thepolylinker of pUX12, a structural gene whose sequence and product areknown, and that lacks its own promoter, was cloned.

For this purpose, a mutant of the tox structural gene carried on theplasmid (Costa, J. J, et al., J. Bacteriol. 148(1):124-130 (1981),Michel, J. L, et al., J. virol. 42:510-518 (1982) which references areincorporated herein by reference) was chosen. The plasmid, pABC402, wastreated simultaneously with the restriction endonucleases ApaI andHindIII (Bishai, W. R, et al., J. Bacteriol. 169:1554-1563 (1987),Bishai, W. R., et al., J. Bacteriol. 169:5140-5151 (1987) whichreferences are incorporated herein by reference). The ApaI site iswithin the structural gene near the N-terminal and the HindIII site liesjust outside of the C-terminal of the tox gene. This 1.2 kb restrictionfragment was separated from the remaining 4.1 kb of the pABC402 vectorusing low melting point agarose.

To create blunt ends for cloning, the tox fragment was treated with T4DNA polymerase. The exonuclease activity of the polymerase cut back theApal 3' ends and the polymerase activity filled in the 5' overhand atthe HindIII site (Maniatis, T. et al., Molecular Cloning, A LaboratoryManual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y. (1982)). Thispurified fragment with blunt ends was ligated into the mixture of pUX12that contains all three reading frames. Individual transformants wererandomly picked and screened by restriction mapping to determine theorientation and reading frame of the inserts. In addition, thenucleotide sequences of the polylinker/adaptor/insert regions weredetermined. All six potential orientation and reading frame combinationswere identified. Finally, extracts from these clones were screened usingWestern blots probed with antisera to diphtheria toxin (Blake, M. S., etal., Anal. Biochem. 136:175-179 (1984), Murphy, J. R., et al., Curr.Topics Microbiol. and Immun. 118:235-251 (1985)).

Reactive toxin related proteins were only detected from clones thatcontained the structural gene in the correct orientation and readingframe. This plasmid is called pUDTAH-1; the DNA sequence of thepolylinker and beginning of the tox structural gene is shown in Table 1.The depicted sequence is the DNA sequence of the beginning of the tox'structural gene in pUDTAH-1. ATG is the start signal for the transcript(lacZ'), GAT begins the modified polylinker of pUX12 and GCC starts thecorrect translational reading frame for the tox' gene.

                                      TABLE 1    __________________________________________________________________________                    SEQUENCES OF PLASMID pUDTAH    __________________________________________________________________________       ATGACCATGATTACGAATTCGAGCTCGCCCGGG                            GATCCATTGTGCTGGAAAG                                          CCACC   SEQ ID NO: 6!    POLYLINKER                            DIPHTHERIALEOTIDE    (ATG = LacZ Translation           ADAPTORS                                              TOX'  GENE    Initiation Codon)    __________________________________________________________________________

EXAMPLE 2 Purification of Chromosomal DNA from Group B Streptococcus

To accomplish the purification of chromosomal DNA from group BStreptococcus chromosomal DNA was isolated from the A909 strain of groupB Streptococcus (Lancefield, R. C., et al., J. Exp. Med. 142:165-179(1975)) by the method of Hull et al., (Hull, R. A., et al., Infect. andImmun. 33:933-938 (1981)) as modified by Rubens et al., (Rubens, C. E.,et al., Proc. Natl. Acad. Sci USA 84:7208-7212 (1987) both of whichreferences are incorporated herein by reference). In brief, mutanolysinwas used to convert the group B Streptococcus strain A909 (Ia/c) straininto protoplasts. The resulting surface extract was found to containnumerous proteins that immunoreact with protective antisera raised tothe intact bacteria. An insoluble protein fraction was partiallypurified using conventional column chromatography. Two fractions,including one which was highly concentrated for a single 14 kilodalton(kd) species, were used to immunize rabbits. Antisera raised againstthese partially purified group B Streptococcus proteins were found to beable to confer passive protection in a mouse virulence assay against aheterologous capsule type of group B Streptococcus which carries the Cproteins.

Group B Streptococcus DNA was purified by centrifugation in abuoyant-density cesium chloride (CsCl) gradient, and the chromosomal DNAwas dialyzed exhaustively against TAE buffer, pH 8.0 (Maniatis, T. etal., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Press,Cold Spring Harbor, N.Y. (1982). The A909 strain of group BStreptococcus has a type 1 capsule, expresses the C proteins and hasbeen used previously in studies of the C proteins (Valtonen, M. V., etal., Microb. Path. 1:191-204 (1986)). It is also the strain of group BStreptococcus that was used in preparing the protective antisera forscreening.

The yield of Group B Streptococcus chromosomal DNA averages 3 to 5 mgfor each 500 ml of an overnight culture of group B Streptococcus. Thepurified DNA was digested separately with 24 commonly used restrictionendonucleases and the resulting fragments were run on a 1.0% agarosegel. A wide range of enzymes were chosen, including those that haveunique sites on the polylinkers commonly used in cloning vectors.Ethidium bromide (EtBr) staining of the gel showed that all of therestriction enzymes yielded a distribution of discrete fragment sizes ofgroup B Streptococcus DNA. This suggests that group B Streptococcus DNAis not modified for any of the restriction enzymes tested.

In order to determine whether there were any inhibitors present to blockligation of the DNA, the restriction endonuclease digestions describedabove were ethanol precipitated, placed in a ligation buffer andincubated overnight at 14° C. with DNA ligase. These samples were againrun on a 1.0% agarose gel and stained with EtBr. The resultingrestriction patterns showed a higher molecular weight distribution.Therefore, there was no inhibition of the ligation of group BStreptococcus DNA.

EXAMPLE 3 Preparation of a Library of Group B Streptococcus ChromosomalDNA

The preparation of a library of group B Streptococcus chromosomal DNA inpUX12 and its transformation into E. coli was performed as follows. Tocleave the group B Streptococcus chromosomal DNA for cloning, fourrestriction enzymes were chosen that give a broad distribution ofrestriction fragment sizes. The pUX12 vector and adaptors are mostefficient when blunt ended fragments are cloned. The enzymes chosenrecognize four base pair sites and leave blunt ends. Group BStreptococcus DNA was partially digested individually with AluI, FunD2,HaeIII and RsaI.

The resulting fragments were mixed, purified with phenol/chloroform,ethanol precipitated and resuspended in a ligation buffer (Maniatis, T.et al., Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Cold Spring Harbor, N.Y. (1982)). One μg of the group BStreptococcus DNA fragments was mixed with 3 μg of the 12-mer and 2 μgof the 8-mer oligonucleotide adaptors. Three microliters of T4 DNAligase (600 units, New England Biolabs), were added and the reaction wasmaintained overnight at 14° C. The free linkers were separated from thegroup B Streptococcus DNA fragments on a potassium acetate velocitygradient (Aruffo, A., et al., Proc. Natl. Acad. Sci. USA 84:8573-8577(1987)).

The pUX12 plasmid containing all three translational reading frames wasdigested with BstXI and the stuffer fragment was removed using a lowmelting point agarose gel. The group B Streptococcus library wasprepared by mixing 10 ng of the adapted group B Streptococcus fragmentswith 100 ng of the linear pUX12 vector in 100 μl of ligation buffer towhich 0.1% T4 DNA ligase was added. The ligation reaction was maintainedovernight at 14° C. and then used to transform the MC1061 strain of E.coli on plates containing ampicillin (Ausubel, F. M., et al., CurrentTopics in Molecular Biology (1987)).

Sixteen of the resulting transformants were isolated, grown overnight inLB and plasmid DNA isolated by mini-preps. The plasmid DNA was digestedwith BamHI, and run on a 1.0% agarose gel. All of the plasmids screenedcontained inserts in the pUX12 vector, and the average insert size was1.4 kb. To date, the plasmid DNA obtained from over 200 clones have beenscreened and only one clone was found that appeared to lack an insert inthe polylinker.

EXAMPLE 4 Characterization of Protective Antisera to be Used inScreening the Library

As discussed earlier, the C proteins have been partially purified by avariety of techniques and protective antisera have been prepared by anumber of investigators (Bevanger, L., et al., Acta Path. Microbiol.Scand. Sect. B. 93:113-119 (1985), Russell-Jones, G. J., et al., J. Exp.Med. 160:1476-1484 (1984), Wilkinson, H. W., et al., Infec. and Immun.4:596-604 (1971)).

A set of experiments was performed to duplicate the work of Valtonen,Kasper and Levy who isolated a 14,000 mw protein from supernatants ofgroup B Streptococcus that elicits protective antibody (Valatonen, M.V., et al., Microb. Path. 1:191-204 (1982) which reference isincorporated herein by reference). This experiment revealed that whenproteolytic inhibitors to the supernatants of group B Streptococcuscultures are added prior to the concentration and purification of the Cproteins (Wong, W. W., et al., J. Immunol. Methods. 82:303-313 (1985)),the 14,000 mw protein was no longer a prominent protein in thesupernatant; This indicated that this protein results from theproteolysis of larger molecular weight C proteins in the supernatants ofgroup B Streptococcus cultures.

EXAMPLE 5 Optimizin Conditions for Screening for Expression in aPlasmid-Based Vector

As discussed above, the most commonly used vectors for the detection ofexpression are based on λgt11 (Young, R. A., et al., Proc. Natl. Acad.Sci. USA 80:1194-1198 (1983)). We were able to increase the sensitivityof detection of expression from the pUX12 plasmid vector by combiningtwo previously described procedures for antibody screening of bacterialcolonies. The transformants from the library were plated overnight andthe resulting colonies transferred to nitrocellulose filters (Bio-Rad).The colonies were lysed by placing the filters in an atmospheresaturated with chloroform (CHCl₃) in a closed container for 30 minutes.The filters were then placed in a lysis buffer and incubated overnightas described by Helfman etal. (Helfman, D. M., etal., Proc. Natl. Acad.USA 80:31-35 (1983)). The antibody screening was done utilizingcommercially prepared E. coli lysate (ratio 1:200) and HorseradishPeroxidase Conjugated, Affinity Purified Goat Anti-Rabbit IgG (ratio1:3000) in the Express-Blot Assay Kit prepared by Bio-Rad Laboratories.By pretreating the colonies with chloroform and the overnight incubationwith DNase and lysozyme described above, it was possible to reduce theratio of primary antibody required from 1:500 to 1:5000.

EXAMPLE 6 Initial Analysis of Positive Clones and their Protein Products

The library of group B Streptococcus chromosomal DNA in the pUX12 vectorwas screened with the above-discussed protective anti-C proteinsantisera. The group B Streptococcus library had an average fragment sizeof 1.4 kb. Transformants were screened as described above, and thensubdloned and rescreened with the antisera three times. Of 20,000 clonesscreened, there were 35 independently isolated clones that reacted withthe protective antisera. The clones were denominated S1-S35, and theplasmids containing the clones were denominated pJMS1-pJMS35. The clonesranged in size from 0.9 to 13.7 kb and have an average size of 4.5 kb.

Plasmid DNA was isolated from the clones by minipreps and the insertssurveyed with four restriction endonucleases. Fourteen of the clones canbe divided into three groups based on sharing identical insert sizes andcommon restriction endonuclease mapping patterns within each group.Clones S1 and S23, discussed below, were found to be members ofdifferent groups.

By further comparing the restriction patterns of the individual clonesit was possible to identify 24 clones that shared common restrictionfragments. Clones S1 and S23 were not found to share any commonrestriction fragments.

Extracts of the clones were prepared, run on Western blots and probedwith the antisera used in screening the library. Six size classes ofprotein antigens were identified (A-F). By combining data from therestriction endonuclease mapping and the Western blots it was possibleto classify 24 of the 35 clones into 6 different protein antigenpatterns (Table 2). This initial classification was done only to get arough survey of the potential number of genes involved. S1 was found tobe 3.5 kd in size, and to belong to antigen protein pattern A. S23 wasfound to be 13.7 kd in size, and to belong to antigen protein pattern D.

                  TABLE 2    ______________________________________       PRELIMINARY CLASSIFICATION OF THE GROUP B     STREPTOCOCCUS                            C PROTEIN CLONES                   Molecular    Protein           Number      Weight  Coding Capacity                                         Size of Antigen    Profile           of clones                     of insert (kb)                                 of DNA insert                                           (in daltons)    ______________________________________        A        6         3.5                                        136,000                                             115,000    B                        1.9                                                 50,000    C                        4.4                                               l30,000    D                        13.7                                        >500,000                                                ll0,000    E                        1.7                                                 50,000    F                        0.9                                                 15,000    ______________________________________

When Western blots of extracts of the clones were probed with antiserato a group B Streptococcus strain that does not express the C proteins,only one group of clones was positive (Protein Profile B). Thisindicates that the majority of positive clones express proteins that areunique to strains that carry the C proteins; these proteins are notcommon to all strains of group B Streptococcus.

EXAMPLE 7 Characterization of the Cloned Gene Sequences

The actual number of C proteins that are expressed by group BStreptococcus has not been determined. Recent immunological studies byBrady et al characterizing C protein typing antisera from the C.D.C.identified four separate antigens (Brady, L. J., et al., J. Infect. Dis.158(5):965-972 (1988)). Preliminary genetic and immunologicalcharacterization of the putative C protein clones of group BStreptococcus suggests that four or five genes encode proteins that arepresent on strains of group B Streptococcus that are known to carry theC proteins. Two groups of experiments were conducted to determinewhether the cloned gene products represent C proteins.

As discussed above, studies of the C proteins had defined twophenotypes: one group of proteins that was sensitive to degradation bypepsin but not trypsin (called TR or α) and another group of proteinsthat was sensitive to degradation by both pepsin and trypsin (called TSor β) (Johnson, D. R., et al., J. Clin. Microbiol. 19:506-510 (1984),Russell-Jones, G. J., et al., J. Exp. Med. 160:1476-1484 (1984)).

The typing antisera, α and β, were used to screen the cloned geneproducts on Western blots (Bevanger, L., et al., Acta Path. Microbiol.Scand. Sect. B. 87:51-54 (1979); Bevanger, L., et al., Acta. Path.Microbiol. Scand. Sect. B. 89:205-209 (1981); Bevanger, L., et al.,Acta. Path. Microbiol. Scand. Sec. B. 91:231-234 (1983); Bevanger, L.,et al., Acta. Path. Microbiol. Scand. Sect. B. 93:113-119 (1985);Bevanger, L., et al., Acta. Path. MicrobioL Immuol. Scand. Sec. B.93:121-124 (1985) which references are incorporated herein byreference).

The α typing sera identified Protein Profile D, and the β typingantisera identified Protein Profile A. These proteins were subjected todigestion with pepsin and trypsin. Protein Profile D is sensitive topepsin but not trypsin, and Protein Profile A is sensitive to bothpepsin and trypsin. These results are consistent with previous studiesand confirm that at least two of the C protein genes have been cloned.

The most important and characteristic property of the C proteins istheir ability to elicit protective antibodies against group BStreptococcus strains that express C proteins. Several approaches couldbe used to prepare antisera against the cloned gene products. Forexample, lysates of the E. coli clones could be directly injected intorabbits in order to determine if the lysates contain proteins capable ofeliciting antibodies to any of the E. coli or group B Streptococcusproteins introduced. The resulting antisera can be preabsorbed with alysate of E. coli prior to testing the antisera to reduce the number ofcross-reacting antibodies. Such a lysate can be used to reduce thenumber of cross-reacting antibodies in both colony blots used forscreening the clones for expression and in Western blots used to studyboth cellular extracts of group B Streptococcus and partially purifiedgroup B Streptococcus proteins.

Representative clones from Protein Profiles A, B and D are sonicated andinjected into rabbits to raise antisera against the cloned group BStreptococcus protein antigens (Lancefield, R. C., et al., J. Exp. Med.142:165-179 (1975), Valtonen, M. V., et al., Microb. Path. 1:191-204(1986)). The control rabbits are injected with E. coli that carriespUX12 without an insert in the polylinker. The antisera is preadsorbedwith an E. coli lysate and screened first on Western blots againstextracts of the clones in the library. Therefore, it is possible todetermine if there are cross-reacting epitopes between the clones and toconfirm that these antisera are directed against the cloned proteinsidentified during the preliminary round of screening.

Alternatively, the preadsorbed antisera may be tested in the mouseprotection model. In this classic model, the mice are injectedintraperitoneally with rabbit antisera (Lancefield, R. C., et al., J.Exp. Med. 142:165-179 (1975)). The following day they are again injectedintraperitoneally with an LD₉₀) of viable group B Streptococcus that areknown to carry C proteins. The endpoint is the death of the mice over a48 hour period.

In order to test the immunogenicity of the proteins expressed by thecloned gene sequences, Escherichia coli cells containing pJMS1 andpJMS23 were grown, and used to prepare cellular extracts. These extractswere then used to immunize rabbits. Antisera raised in response toimmunization with the S1 and the S23 extracts were tested using themouse protection model.

When the mouse protection model experiment was performed, the antiseraraised from the clones representing Protein Profiles A and D (S1 andS23, respectively), were each found to be protective. Antisera from aclone representing Protein Profile C was not protective and the controlantisera also did not show protection. The antisera raised against theclones expressing Protein Profile C also binds to proteins extractedfrom strains of group B Streptococcus that do not carry the C protein.Therefore, this group of clones do not encode C proteins. In summary,five of the six groups of clones do not encode proteins that are uniqueto strains of group B Streptococcus that express C proteins.

The initial biochemical, immunological and functional analysis of two ofthe groups of clones demonstrates that at least two C proteins genes (S1and S23) have been successfully cloned. This is the first demonstrationthat single polypeptide gene products cloned from group B Streptococcuscan elicit protective immunity. Antibodies to S1 were found to be ableto bind two bands of the A909 extract at 50 and 60 kd. Antibodies to S23were found to be able to bind to a regularly repeating pattern of bandsin the group B Streptococcus surface extract which ranged in MWfrom >180 kd to 40 kd. A monoclonal antibody derived from the A909extract showed this same repeating pattern of immunoreactivity. Thisindicates that a single epitope was recognized in different molecularweight proteins and suggests a regularly repeating structure. Theproteins recognized by the S1 antiserum were susceptible to pepsin andtrypsin degradation whereas those recognized by the S23 antiserum weresusceptible to pepsin but not to trypsin. This experiment shows thatthese proteins partially purified from group B Streptococcus andexpressed from the group B Streptococcus cloned genes represent thealpha and beta antigens of the C protein of group B Streptococcus.

The 35 potential C protein clones described above may be evaluated bothgenetically and immunologically to determine the number of genes thatare present. In addition, the isolation of these clones permits thegenes which confer protective immunity to group B Streptococcusinfection may be identified. It is likely that the protective antiseraused to obtain the initial clones also detected proteins other than theC proteins. The use of such other proteins in a therapy againstStreptococcus B infection is also contemplated by the present invention.Since a major goal of the present invention is the isolation andidentification of the proteins involved in immunity, antisera preparedagainst the proteins expressed by these clones may be studied in themouse protection model. Those genes that express proteins that areprotective are preferred proteins for a conjugate vaccine.

As discussed above, the initial screening of group B Streptococcuschromosomal DNA in an E. coli/pUX12 vector library with protectiveantisera resulted in 35 independently isolated clones. By combining datafrom restriction endonuclease mapping of the cloned fragments andWestern blots of protein extracts from the clones, it was possible totentatively classify 24 of the 35 clones into 6 different proteinantigen patterns (Table 2). This survey permitted a determination of thepotential number of genes isolated.

To further characterize such clones, colony blots are preferably used todetermine which clones share common DNA sequences. For such blots, asingle colony of each of the clones is placed in a well of microtiterdish containing LB broth and grown at 37° C. overnight. Control coloniesinclude the host E. coli strain and the E. coli strain containing pUX12.The overnight cultures are transferred onto a nitrocellulose filter onan agar plate containing the same culture medium. These plates are grownup over 8 hours at 37° C. and the nitrocellulose filter containing thefreshly grown colonies is prepared to be screened for DNA-DNAhybridization. The probes are prepared from the group B StreptococcusDNA inserts in the pUX12 library. Mini-preps are used to obtain plasmidDNA from the clones. The polylinker in pUX12 has both a BamHI and BstXIsite on either side of the insert; therefore, the group B Streptococcusinsert is excised from the plasmid using either BamHI or BstXI.Fortunately, the chromosomal DNA of group B Streptococcus contains fewBamHl sites and many of the inserts are removed from the vector in onefragment as the result of digestion with BamHI. Low melting pointagarose is used to separate the plasmid vector from the inserts. Theinserts will be cut from the agarose gel and directly labeled by randomprime labeling. The labeled inserts are then used to probe the colonyblots. This results in the identification of clones that share DNAsequences.

Thus, on the basis of the information obtained from the colony blotsdescribed above, the 35 clones are placed into groups that share DNAsequences. These groups are mapped with multiple restrictionendonucleases to determine the relationship of each clone to the otherswithin that region of the DNA. Since the host plasmid, pUX12, containsmany unique restriction endonucleases sites that are present only in thepolylinker, much of the restriction mapping can be done utilizing theplasmid mini-prep DNA without needing to purify the inserts separately.By combining the colony blot data with detailed restriction mapping itis possible to get a reasonable assessment of the number of genetic lociinvolved. If some of the groups of clones do not represent the genes ofinterest in their entirety, it may be necessary to use these clones toisolate other more complete copies of the genes from the chromosomallibrary. However, given the large average size distribution of theinitial 35 clones isolated, it is likely that some may represent acomplete open reading frame.

Before proceeding with a genetic analysis, antisera is preferablyprepared against the cloned gene products, and utilized in the mouseprotection model to determine the ability of these antisera to protectagainst infection with group B Streptococcus (Lancefield, R. C., et al.,J. Exp. Med. 142:165-179 (1975), Valtonen, M. V., et al., Microb. Path.1:191-204 (1986)).

A clone whose expressed protein is able to elicit protective antibodiesis a preferred candidate for use in a conjugate vaccine. Clones whoseexpressed protein fails to elicit protective antibodies may be furtheranalyzed to determine whether they are also candidates for a vaccine.Since the C proteins are membrane associated, a failure of proteinexpressed by a clone to elicit protective antibodies may reflect thefact that the protein may not be stable in E. coli, and in a high copynumber vector. This problem has occurred in cloning other membraneproteins from both group A and group B Streptococcus (Kehoe, M. et al.,Kehoe, M., et al., Infect. and Immun. 43:804-810 (1984), Schneewind, O.,et al., Infect. and Immun. 56:2174-2179 (1988)). Several of the 35clones isolated in the preliminary studies show a small colonymorphology. In addition, some of these clones are unstable and have beenfound to delete part of the group B Streptococcus DNA insert from thepUX12 polylinker. There are several techniques that can be used tostabilize these clones including: cloning into a low copy number vectoror behind a promoter that can be down-regulated, growing the clones at30° C. instead of 37° C., cloning into a vector that has been adapted toaccumulate membrane proteins. In addition, it is possible to transformthe plasmids into an E. coli host, pcnB, that restricts the copy numberof pBR322 derived plasmids like pUX12 (Lopilato J., et al., Mol. Gen.Genet. 205:285-290 (1986) which reference is incorporated herein byreference).

A failure of a clone to express protein which elicits protectiveantibodies may also indicate that the expressed protein lacks an epitopewhich is important for protection. This could be the case if the entiregene was not cloned or could not be expressed in E. coli. It might alsobe problem if there is post-transcriptional processing of the C proteinsin group B Streptococcus but not for the cloned C protein genes in E.coli. It might be necessary either to subclone out the complete geneand/or transfer it into an alternate host background where it can beexpressed.

A failure of a clone to express protein which elicits protectiveantibodies may also indicate that antibodies elicited from antigensproduced in Eschefichia coli may differ from those elicited from anStreptococche native C proteins on group B Streptococcus. In addition,the lysed bacterial extracts used to immunize the rabbits contain anumber of E. coli protein antigens. Therefore, it may be necessary toobtain antisera for testing in the animal model from partially purifiedgene products instead of from the entire organism.

Any cloned group B Streptococcus proteins that are able to elicitprotective antibodies can be called C proteins. The antisera preparedfor this group of experiments will also be used for localizing theseprotein.

EXAMPLE 8 Mapping, Characterization and Sequencing of the C ProteinGenes

In order to further characterize the C protein genes, a fine structuregenetic map of C protein gene clones described above may be prepared andtheir DNA sequence(s) determined. Such mapping is preferablyaccomplished utilizing genomic Southern blots. By determining the DNAsequences of the C protein genes, one can determine the structure of thegenes including their ribosomal binding sites, potential promoters,signal sequences, and any unusual repetitive sequences. The DNAsequences are preferably compared to a library of known DNA sequences tosee if there is homology with other genes that have been characterized.In addition, the protein sequences of the C proteins can be determinedfrom DNA sequences of their genes. It is often possible to makepredictions about the structure, function and cellular location of aprotein from the analysis of its protein sequence.

Genomic Southern blots are, thus, preferably used to determine if any ofthe genes are linked. For this technique, group B Streptococcuschromosomal DNA is digested individually with several differentrestriction endonucleases that identify sequences containing six or morebase pairs. The purpose is to obtain larger segments of chromosomal DNAthat may carry more than one gene. The individual endonucleasedigestions are then run out on an agarose gel and transferred ontonitrocellulose. The Southern blots are then probed with the labelledinserts derived from the above-described library. If two clones that didnot appear related by the colony blots or endonuclease mapping bind tosimilar chromosomal bands, this would indicate that either they are partof the same gene, or that they are two genes that are closely linked onthe chromosome. In either case, there are several ways to clone outthese larger gene segments for further study. One technique is toprepare a cosmid library of group B Streptococcus and screen forhybridization with one of the probes of interest. When a clone isobtained that contains two or more genes of interest it could beendonuclease mapped and studied for the expression of protectiveantigens as described for the previously described clones.

The identification of the above-described clones permits their DNAsequences to be determined. If the clones are on the pUX12 plasmid, itis possible to use double stranded DNA sequencing with reversetranscriptase to sequence from oligonucleotide primers prepared to thepolylinker. This technique was used earlier in characterizing the pUX12plasmid and is a rapid way to sequence multiple additionaloligonucleotide primers to sequence a gene that is larger than 600 basepairs. Therefore, the DNA sequencing for the C protein genes ispreferably performed by subcloning into an M13, single stranded DNAsequencing system (Ausubel, F. M., et al., Current Topics in MolecularBiology (1987)).

The elucidation of the DNA sequences of the C proteins providessubstantial information regarding the structure, function and regulationof the genes and their protein products. As discussed earlier, theheterogeneity in the sizes of C proteins isolated by many investigatorsand their apparent antigenic diversity suggests the possibility ofeither a gene family, or a post-transcriptional mechanism for modifyingthe protein products of the C protein genes (Ferrieri, P., et al.,Infect. Immun. 27:1023-1032 (1980)). The M protein of group AStreptococcus was discussed earlier as an example of this phenomenon(Scott, J. R., et al., Proc. Natl. Acad. Sci. USA 82:1822-1826 (1985)).Although the DNA sequence of M protein shows no homology with group BStreptococcus chromosomal DNA by hybridization, there may be structuralhomologies between their DNA sequences (Hollingshead, S. K., et al., J.Biol. Chem. 261:1677-1686 (1986), Scott, J. R., et al., Proc. Natl.Acad. Sci. USA 82:1822-1826 (1985), Scott, J. R., et al., Infect. andImmun. 52:609-612 (1986)). The DNA sequences of the C proteins arepreferably compared with a library of known DNA sequences. In addition,the amino acid sequences derived from the DNA sequences are comparedwith a library of known amino acid sequences.

EXAMPLE 9 Prevalence of the C Protein Genes

To determine the prevalence of the C protein genes, chromosomal DNA fromclinical and laboratory isolates of the various serotypes of group BStreptococcus are probed on genomic Southern blots with the C proteingenes. In addition, comparison of the phenotypic expression asdetermined by precipitin techniques with genetic composition as shown byDNA-DNA hybridization is preformed in order to provide informationregarding the regulation of expression of the C protein genes. Theprobes of the C protein genes are used to screen chromosomal DNA fromother types of Streptococcus, and other bacterial pathogens.

Probes are prepared and labelled from the C protein genes of isolates ofgroup B Streptococcus which includes most of the original typing strainsused by Lancefield (Lancefield, R. C., et al., J. Exp. Med. 142:165-179(1975)). Colony blots of the 24 clinical and laboratory isolates ofgroup B Streptococcus are screened using the microtiter techniquedescribed above. The ability of the various strains to hybridize to theC protein genes is then compared with the phenotypic characteristics ofthese organisms in binding to typing antisera directed against the Cproteins. In this manner, it is possible to determine what strains carryany or all of the C protein genes, and whether some strains carry silentor cryptic copies of these genes.

Those strains that hybridize to the C protein gene probes on colonyblots are then screened using genomic Southern blots to determine thesize, structure and location of their C protein genes. Chromosomal DNAisolated from the strains of group B Streptococcus that show binding onthe colony blots is digested with restriction endonucleases, run on anagarose gel and blotted onto nitrocellulose. These Southern blots areprobed with probes of the C protein genes. In this manner, it ispossible to determine if there are differences in the location and sizeof these genes in the different serotypes of group B Streptococcus andto compare clinical (i.e. potentially virulent) isolates with laboratorystrains (and with those which colonize clinically but are not associatedwith infection).

The C protein gene probes are also preferably used to screen otherstreptococcal strains and a variety of pathogenic bacteria.Streptococcal strains are known to share other proteins associated withvirulence including the M and G proteins (Fahnestock, S. R., et al., J.Bact. 167(3):870-880 (1986), Heath, D. G., et al., Infec. and Immun.55:1233-1238 (1987), Scott, J. R., et al, Infec. and Immun. 52:609-612(1986), Walker, J. A., et al., Infec. and Immun. 55:1184-1189 (1987)which references are incorporated herein by reference). The strains tobe tested are first screened using colony blots to determine whetherthey have any homologous sequences with the C protein genes probes.Genomic Southern blots are then prepared with the chromosomal DNA of thebacterial strains that test positive on the colony blots. These blotsare then probed with the C protein genes to localize and define theareas of homology, such as a region of a C protein which serves as amembrane anchor, binds to the Fc region of immunoglobulins, or sharesregions of homology with other genes with similar functions in otherbacteria.

EXAMPLE 10 Modification of the C Protein Genes in Group B STREPTOCOCCUS

A number of potential virulence associated properties have been ascribedto the C proteins including resistance opsonization and inhibition ofintracellular killing following phagocytosis (Payne, N. R, et al., J.Infec. Dis. 151:672-681 (1985), Payne, N. R., et al., Infect. and Immun.55:1243-1251 (1987)). To better understand the roles of the C proteinsin virulence, isogeneic strains are constructed in which the C proteingenes are individually mutated. These strains will be tested forvirulence in the neonatal rat model (Zeligs, B. J., et al., Infec. andImmun. 37:255-263 (1982). Two methods may be utilized to createisogeneic strains to evaluate the role of the C proteins in thevirulence of group B Streptococcus. Preferably, tranposon mutagenesiswith the self-conjugative transposon tn916 may be employed.Alternatively, site-directed mutagenesis may be used. The lack ofefficient methods for genetic manipulation in group B Streptococcusnecessitates the development of new genetic techniques to modify genesin group B Streptococcus and create isogeneic strains for studyingvirulence (Lopilato, J., et al., Mol. Gen. Genet. 205:285-290 (1986)which reference is incorporated herein by reference).

Transposon insertional mutagenesis is a commonly used technique forconstructing isogeneic strains that differ in the expression of antigensassociated with virulence, and its use in group B Streptococcus is welldescribed (Caparon, M. G., et al., Proc. Natl. Acad. Sci. USA84:8677-8681 (1987), Rubens, C. E., et al., Proc. Natl. Acad. Sci USA84:7208-7212 (1987), Wanger, A. R, Res. Vet. Sci. 38:202-208 (1985),Weiser, J. N., Trans Assoc. Amer. Phys. 98:384-391 (1985) whichreferences are incorporated herein by reference). Rubens, et aL havedemonstrated the utility of Tn916 in studies of the group BStreptococcus capsule (Rubens, C. E., Proc. Natl. Acad. Sci. USA84:7208-7212 (1987). The self-conjugating transposon TN916 may be madefrom Streptococcus faecalis into group B Streptococcus as previouslydescribed (Wanger, A. R., Res. Vet. Sci. 38:202-208 (1985) whichreference is incorporated herein by reference). Strains are selected forthe acquisition of an antibiotic resistance marker, and screened oncolony blots for the absence of expression of the C proteins as detectedby the specific antisera prepared as described above. Isolates that donot appear to express the C proteins can be further mapped using genomicSouthern blots to localize the insertion within the C protein genes. Theoriginal Tn916 strain carried tet^(R) ; however, an erythromycinresistance marker has recently been cloned into Tn916 (Rubens, C. E., etal., Plasmid 20:137-142 (1988)). It is necessary to show that, followingmutagenesis with Tn916, only one copy of the transposon is carried bythe mutant strain and that the transposon is localized within the Cprotein gene.

The application of these techniques to deleting the C protein genes ingroup B Streptococcus is straightforward, unless a C protein genes isessential to the survival of group B Streptococcus. However, strains ofgroup B Streptococcus have been described that lack any detectable Cprotein and it is unusual for a bacterial virulence determinant to be anessential gene for survival in vitro. A n additional use of Tn916 thatwill be explored is the identification of potential regulatory elementsof the C protein genes.

In the event that specific defined mutations are desired or if the Cprotein gene is essential for the viability of group B Streptococcus,techniques of site-directed mutagenesis may be employed (for example toproduce conditional mutants). Site-directed mutagenesis may thus be usedfor the genetic analysis of group B Streptococcus proteins. One problemthat has delayed the development of these techniques in group BStreptococcus is the difficulty encountered in transforming group BStreptococcus. Electroporation has proven valuable in introducing DNAinto bacteria that are otherwise difficult to transform (Shigekawa, K.,et al., BioTech. 6:742-751 (1988) which reference is incorporated hereinby reference). Conditions for transforming group B Streptococcusutilizing electroporation may be utilized to surmount this obstacle. Itis thus possible to do site directed mutagenesis, to evaluatecomplementation, and to introduce C protein genes into group BStreptococcus strains that do not express the C proteins. Any of severalapproaches may be utilized to insert native or mutated C protein genesinto strains of group B Streptococcus. For example, a drug resistancemarker may be inserted within the C protein gene clones in pUX12. A drugresistance marker that can be expressed in group B Streptococcus, butthat is not normally present, is preferred. This modified pUX12 proteinclone is transformed into group B Streptococcus using electroporation(Shigekawa, K., et al., BioTech 6:742-751 (1988) which reference isincorporated herein by reference). Since the pUX12 plasmid cannotreplicate in group B Streptococcus, those strains that acquire the drugresistance phenotype would likely do so by homologous recombinationbetween the C protein gene on the host GB chromosome and the mutated Cprotein carried on the pUX12 plasmid. The mutants are screened asdescribed above. If there are no homologous sequences in the recipientstrain, it is possible to construct a vector with the C protein geneinserted within a known streptococcal gene, i.e., a native drugresistance marker gene from group B Streptococcus. Followingelectroporation, such a plasmid construct would integrate into thechromosome via homologous recombination.

Alternatively, modified C protein genes could be introduced into thegroup B Streptococcus chromosome by inserting the genes into theself-conjugating transposon Tn916 and introducing the modifiedtransposons via mating from Streptococcusfaecalis. This technique wasused to successfully modify Tn916 with an erythromycin gene and insertthis gene into the chromosome of group B Streptococcus (Rubens, C. E.,et al., Plasmid 20:137-142 (1988)). It is necessary to show that,following mutagenesis with Tn916, only one copy of the transposon iscarried by the mutant strain and that the transposon is localized withinthe C protein gene.

EXAMPLE 11 Evaluation of the Role of the C Proteins in Virulence ofGroup B STREPTOCOCCUS

Previous studies that compared strains of group B Streptococcus that doand do not carry C proteins involved isolates that were not known to beisogeneic (Ferrieri, P., et al., Rev. Inf. Dis. 10(2):1004-1071 (1988)).Therefore, it was not possible to determine whether the differences invirulence observed are related to the C proteins or to some othervirulence determinant. The construction of isogeneic strains havingeither intact C protein genes or C protein gene deletions permit acharacterization of the role of the C protein in virulence. The strainsare preferably tested in the neonatal rat model for virulence and in themouse protection model for their immunological properties. A secondimportant test of virulence is the ability of a gene to restorevirulence through reversion of allelic replacement in a mutant strain.By inserting the C protein genes into group B Streptococcus strains thateither do not carry the gene or which carry inactivated C protein genes,it is possible to determine the effect of the C protein by examining thevirulence of the resulting construct in the above animal models.

Isogeneic strains of group B Streptococcus in which the C protein genesare individually mutated may be created using either transposonmutagenesis or site-directed mutagenesis. Such strains are preferablycharacterized on genomic Southern blots to determine that only a singleinsertion is present on the chromosome. The location of these insertionsmay be ascertained using the fine structure genetic mapping techniquesdiscussed above. The isogeneic strains are then tested for virulence inthe neonatal rat model (Zeligs, B. J., et al., Infec. and Immun.37:255-263 (1982)).

Transposon mutagenesis permits the identification of genes involved inregulating the expression of the C proteins. For example, strainscarrying the wild type C protein genes which are found to no longerexpress C proteins following transposon mutagenesis and in whichtransposon is not located within the C protein structural gene, carrymutations in sequences involved in the regulation of expression of the Cprotein genes. This approach was used successfully in characterizing themry locus in group A Streptococcus that is involved in regulation of theM protein (Caparon, M. G., et al., Proc. Natl. Acad. Sci. USA84:8677-8681 (1987), Robbins, J. C., et al., J. Bacteiol. 169:5633-5640(1987) which references are incorporated herein by reference). Suchmethods may also be used to produce strains which overexpresses the Cproteins, or which produce C proteins of altered virulence or immunity.

EXAMPLE 12 Localization of the C Proteins on Group B Streptococcus andEvaluation of their Ability to Bind to Immunoglobulins

Lancefield and others have shown that antibody to the C proteins bindsto the outer membrane of group B Streptococcus (Lancefield, R. C., etal., J. Exp. Med. 142:165-179 (1975), Wagner, B., et al., J. Gen.Microbiol. 118:95-105 (1980)). This suggests that the C protein is anouter membrane protein. C proteins can also be isolated from thesupernatants of cultures of group B Streptococcus, indicating that theseproteins may be either secreted by group B Streptococcus or lost at ahigh rate from the cell surface. The DNA and protein sequences derivedfrom the C protein genes are valuable in determining the structure andfunction of the C proteins. One potential virulence determinant commonlydescribed for the C proteins is the ability to bind to theimmunoglobulin, IgA (Ferrieri, P., et al., Rev. Inf. Dis.10(2):1004-1071 (1988), Russell-Jones, G. J., et al., J. Exp. Med.160:1467-1475 (1984)).

Immuno-electron microscopy has been utilized to localize cell surfacedeterminants that are detected by specific antibody. Antisera raisedagainst the C protein clones of group B Streptococcus is incubated withgroup B Streptococcus strains that carry the C proteins.Ferritin-conjugated goat anti-rabbit IgG is used to detect the antigenon the cell surface as previously described (Rubens, C. E., et al.,Proc. Natl. Acad. Sci. USA 84:7208-7212 (1987), Wagner B., et al., J.Gen. Microbiol. 118:95-105 (1980)).

A simple determination of the ability of C proteins to bind toimmunoglobulins can be assessed using Western blots. Cellular extractsof both the E. coli clones containing the C protein genes and of group BStreptococcus strains that carry the C proteins can be run on SDS-PAGEand blotted onto nitrocellulose. Controls include extracts of E. colicarrying the wild type pUX12 plasmid, strains of group B Streptococcusthat do not carry the C protein genes, and isogeneic group BStreptococcus strains in which the C protein genes have beeninactivated. The Western blots can be probed individually with labelledimmunoglobulins, e.g., IgG, IgM, IgA, and their components, e.g., the Fcor F(ab)₂ fragments (Heath, D. G., et al., Infect. and Immun.55:1233-1238 (1987), Russell-Jones, G. J., et al., J. Exp. Med.160:1467-1475 (1984)). The immunoglobulins are preferably iodinatedusing either iodogen or chloramine T.

A more specific way to measure the ability of the C proteins to bind toimmunoglobulins and their components involves purifying the C proteinsand using them directly in a binding assay (Fahnestock, S. R., et al.,J. Bact. 167(3):870-880 (1986), Heath, D. G., et al., Infect. and Immun.55:1233-1238 (1987)). Using the protein sequence, one can purify the Cprotein. In addition, since it is possible to express the C proteingenes in E. coli, one may construct E. coli strains that overproduce theC proteins and thereby obtain larger amounts of C proteins forpurification.

EXAMPLE 13 Use of the Cloned C Protein Antigenes of Group BStreptococcus Vaccine

The above-described protective C protein antigens of group BStreptococcus were tested for their potential in a conjugate vaccine. Toassess this potential, cellular extracts of E. coli containing pJMS 1 orpJMS23 were prepared as described above, and used to immunize rabbits.The resulting antisera was tested in the mouse lethality model for itsability to protect mice from infection by the group B Streptococcusstrain H36B. Strain H36B carries the C protein of group B Streptococcus.As a control, the ability of the antisera to protect the mice againstinfection by Streptococcus strain 515 (which does not carry the Cprotein) was determined. The results of this experiment are shown inFIG. 4.

EXAMPLE 14 The Sequence of the C Protein Alpha Antigen and its RepeatingUnits

As stated above, Streptococcus agalactine group B Streptococcus (GBS)!is an important pathogen in neonatal sepsis and meningitis, postpartumendometritis, and infections in adults, in particular in diabetics andimmunocompromised hosts (Baker, C. J., et al., in Infectious Diseases ofthe Fetus and Newborn Infant, Remington, J. S. et al. Saunders,Philadelphia, (1990) pp. 742-811)). The best-studied GBS virulencedeterminants are the type-specific capsular polysaccharides that areessential for pathogenesis (Rubens, C. E., et al., Proc. Natl. Acad.Sci. USA 84:7208-7212 (1987); Wessels, M. R., et al., Proc. Natl. Acad.Sci. USA 86:8983-8987 (1989)). The roles of GBS surface proteins ininfection are less well understood (Ferrieri, P. Rev. Infect. Dis.S363-S366 (1988); Michel, J. L., et al. in Genetics and MolecularBiology of Streptococci, Lactococci, and Enterococci, Dunny, G. M. etal. eds., Am. Soc. Microbiol., Washington (1991), pp. 214-218). The Cproteins are surface-associated antigens expressed by most clinicalisolates of capsular types. Ia, Ib, and II and are thought to play arole in both virulence and immunity (Johnson, D. R., et al., J. Clin.Microbiol. 19:506-510 (1984); Madoff, L. C., et al. Infect. Immun.59:2638-2644 (1991)). Two C protein antigens, alpha and beta, have beendescribed biochemically and immunologically (Michel, J. L., et al. inGenetics and Molecular Biology of Streptococci, Lactococci, andEnterococci, Dunny, G. M. et al. eds., Am. Soc. Microbiol., Washington(1991), pp. 214-218).

In 1975, Lancefield et al. (Lancefield, R. C., et al., J. Exp. Med.142:165-179 (1975)) showed that antibodies raised to the C proteins inrabbits protected mice challenged with GBS bearing the C proteins. Amonoclonal antibody to the alpha antigen (4G8) that induces opsonickilling of GBS and protects mice from lethal challenge with GBS has beendescribed (Madoff, L. C., et al., Infect. Immun. 59:204-210 (1991),incorporated herein by reference). As shown above, the gene encoding theencoding alpha and beta antigens were cloned and expressed inEschefichia coli. It was shown that antibodies raised to the clones ofboth alpha and beta encode different C proteins that define uniqueprotective epitopes (Michel, J. L., et al., Infect. Immnun. 59:2023-2028(1991)). The alpha and beta antigens are independently expressed andantigenically distinct proteins.

The C protein beta antigen that specifically binds to human serum IgAhas been cloned (Michel, J. L., et al., Infect. Immun. 59:2023-2028(1991); Cleat, P. H., et al., Infect. Immun. 55:1151-1155 (1987)) andsequenced (Heden, L.-O., et al., Eur. J. Inununol. 21:1481-1490 (1991);Jerlstrom, P. G., et al., Mol. Microbiol. 5:843-849 (1991)). However,the role of the beta antigen and IgA binding in virulence is not known.Studies by Ferrieri et al. (Payne, N. R., et al., J. Infect. Dis.151:672-681 (1985); Payne, N. R., et al., Infect. Immnun. 55:1243-1251(1987)) showed that C protein-bearing strains of GBS resist phagocytosisand inhibit intracellular killing. Opsonophagocytic killing in thepresence of alpha antigen-specific monoclonal antibody (4G8) correlateddirectly with increasing molecular mass of the alpha antigen and withthe quantity of alpha antigen expressed on the bacterial cell surface(Madoff, L. C., et al., Infect. Immun. 59:2638-2644 (1991)). GBS strainsexpressing the alpha antigen Were resistant to killing bypolymorphonuclear leukocytes in the absence of specific antibody;however, this resistance was not dependent on the size of the alphaantigen.

The completed nucleotide sequence of bca and flanking regions reportedhere provides information regarding the size, structure, and compositionof the alpha antigen gene. An interesting feature of both the native andcloned gene products of the alpha antigen is that they exhibit proteinheterogeneity by expressing a regularly repeating ladder of proteinsdiffering by approximately 8000 Da (Madoff, L. C., et al., Infect.Immun. 59:204-210 (1991); Michel, J. L., et al., Infect. Immun.59:2023-2028 (1991)). Since the protective monoclonal antibody 4G8 bindsto the repeat region, this region defines a protective epitope (Madoff,L. C., Infect. Immun. 59:2023-2028 (1991)). Smaller tandemly repeatedsequences encoding immunodominant epitopes have been reported in anumber of pathogens but have not been associated with the proteinheterogeneity seen in the alpha antigen (Enes, V., et al., Science225:628-630 (1984); Fischetti, V. A., et al., Rev. Infect. Dis.S356-S359 (1988); Pereira, M. E., et al., J. Exp. Med. 174:179-191(1991); Fischetti, V. A., et al., in Genetics and Molecular Biology ofStreptococci, Lactococci, and Enterococci, Dunny et aL eds. Am. Soc.Microbiol., Washington (1991), pp. 290-294; Dailey, D. C., et al.,Infect. Immnun. 59:2083-2088 (1991); vonEichel-Streiber, C., et al.,Gene 96:107-113 (1990)). Though the maximum molecular size of the alphaantigen differs among strains of GBS, this protein heterogeneity is aconstant feature (Madoff, L. C., et al. Infect. Immun. 59:2638-2644(1991)).

The nucleotide sequence of bca contains nine identical 246-nucleotidetandem repeating units. The estimated size of the peptide encoded byeach of these repeats is 8665 Da and correlates with the intervals foundin the heterogeneous laddering of the alpha antigen. The amino acidsequence derived from the DNA sequence revealed both significanthomologies and important differences between the alpha antigen and otherstreptococcal proteins (Heden, L. O., et al., Eur. J. Immunol.21:1481-1490 (1991); Jerlstrom, P. G., et al., Mol. Microbiol. 5:843-849(1991); Fischetti, V. A., et al., in Genetics and Molecular Biology ofStreptococci, Lactococci, and Enterococci, Dunny et al. eds. Am. Soc.Microbiol., Washington (1991), pp. 290-294). The repeating units of thealpha antigen suggest possible mechanisms for phenotypic and genotypicvariability and provide natural sites for gene rearrangements that couldgenerate antigen diversity.

Materials and Methods

Bacterial Strains, Plasmids, Transposons, and Media.

GBS strain A909 (type 1a/C.sub.α,β) (Lancefield, R. C., et al., J. Exp.Med. 142:165-179 (1975)), E. coli strains MC1061 and DK1 (Ausubel, F.M., et al., Current Protocols in Molecular Biology, Wiley, New York(1990)), pCNB (Lopilato, J. et al., Mol. Gen. Genet. 205:285-290(1986)), DH5α (a derivative of DH1; GIBCO/BRL), and NK-8032; E. coliplasmids and clones pUC12, pUX12, and pJMS23; and the transposon Tn5seq1have been described (Michel, J. L., et al., Infect. Immun. 59:2023-2028(1991)). The plasmid pGEM-7Zf(-) was purchased from Promega, Madison,Wis., USA. Additional subclones of pJMS23 (pJMS23-1, -7, -9, and -10)are described below. Growth media for GBS and E. coli and antibioticsfor selection have been described (Michel, J. L., et al., Infect. Immun.59:2023-2028 (1991)).

DNA Procedures and Nucleotide Sequencing Strategy.

Standard procedures for the preparation of plasmid DNA, synthesis andpurification of oligonucleotides, restriction endonuclease mapping,agarose gel electrophoresis, and Southern blot hybridization are fromAusubel et al. (Ausubel, F. M., et al., Current Protocols in MolecularBiology, Wiley, New York (1990)). Restriction endonucleases and otherenzymes for manipulation of DNA (e.g., DNase, RNase, and ligase) wereobtained from New England Biolabs and Boehringer Manneheim. Transposonmutagenesis utilized lambda-Tn5seq1 (Nag, D. K., et al., Gene 64:135-145(1988)).

Nucleotide sequencing of double-stranded DNA used plasmids containingtransposon Tn5seq1 insertions using primers of Sp6 or T7 promoters forbidirectional sequencing, synthetic oligonucleotide primers, and nesteddeletions using Erase-a-Base (Promega, Madison Wis., USA; Henikoff, S.,Gene 28:351 (1984)). A total of 12 primers were prepared to obtain thesequence in both directions for the areas of the gene flanking therepeat region. Sequencing of the region of repetitive DNA was completedwith exonuclease III-generated nested deletions. All sequencing employedSequenase kit, version 2, used according to manufacturer'sspecifications for double-stranded sequencing (United StatesBiochemical). Adenosine 5'- α- ³⁵ S!thio!triphosphate was obtained fromAmersham. GenAmp PCR kit with AmpliTaq polymerase was used according tomanufacturer's instructions (Perkin-Elmer/Cetus).

Subclones pJMS23-1, pJMS23-7, and pJMS23-10 were prepared for transposonmutagenesis to target smaller regions within bca (Michel, J. L. et al.,Infect. Immun. 59:2023-2028 (1991)). Subclone pJMS23-1 contains a5.9-kilobase HindIII fragment in pUX12; pJMS23-7 contains 2.8-kilobaseAlu I fragment from pJMS23-1 ligated into the Hincll site in thepolylinker of pUC12; and pJMS23-10 is a BsaB1/Sma I double restrictionendonuclease digestion of pJMS23-7 that yielded a 2.3 kilobase insertcontaining the repeat region. For nested deletions the Alu I fragmentfrom pJMS23-1 was ligated into the Sma I site on pGEM-7Zf(-) to createpJMS23-9. Nested deletions were constructed in the forward directionfrom the HindIII and Nsi I sites and in the reverse direction from EcoRIand Sph I sites. The sizes of the subclones, mutants, and deletions usedfor sequencing were confirmed by restriction endonuclease mapping and/orPCR with primers to the pUC12 polylinker and to Tn5seq1 (Sp6 and T7).

Data analysis used the Department of Molecular Biology computer atMassachusetts General Hospital (Boston) with Genetics Computer Group(Madison, Wis.) version 7 software and the BLAST network of the NationalCenter for Biotechnology Information of the National Institutes ofHealth (Bethesda, Md.).

Monoclonal Antibodies, SDS/PAGE, and Western Immunoblots.

Extracts of GBS and E. coli proteins, SDS/PAGE, immunoblotting, andprobing with the alpha antigen monoclonal antibody 4G8 were performed asdescribed in Madoff, L. C., et al. Infect. Immun. 59:2638-2644 (1991),in Madoff, L. C., et al., Infect. Immun. 59:204-210 (1991), and inMichel, J. L., et al., Infect. Immun. 59:2023-2028 (1991).

Results

Nucleotide Sequence of bca.

Subdlones of pJMS23, which encodes the bca locus from GBS strain A909(type Ia/C) and expresses the alpha antigen in E. coli, were used fordetermining the sequence of bca (Michel, J. L., et al., Infect. Immun.59:2023-2028 (1991)). As is often the case with Gram-positive genescloned into E. coli, many of the subclones were unstable (Schneewind,O., et al., Infect. Immun. 56:2174-2179 (1988)). This problem iscompounded in bca by a large region of repetitive DNA that providesmultiple, fixed sites for homologous recombination.

Homologous recombination such as this may be purposely taken advantageof to generate a population of recombinant hosts that express a varietyof alpha antigen functional derivatives. Such a population would be amixtures of the alpha antigens and their functional derivatives and maybe utilized in the vaccines of the invention to provide a wide range ofalpha antigen sequences against which the host may direct the immuneresponse.

To verify that pJMS23 encodes the complete native gene withoutdeletions, Southern blots of genomic DNA from A909 were probed with genefragments from the clone. There were no differences found in therestriction maps of bca between A909 and pJMS23. The complete nucleotidesequence of bca was obtained independently on both stands using threestrategies: transposon mutagenesis with Tn5seq1, syntheticoligonucleotide primers, and exonuclease III nested deletions (FIG. 5).

The complete nucleotide sequence of the bca locus and derived amino acidsequence for a single, large open reading frame are shown in FIG. 6A-6C.The structural gene consists of 3063 nucleotides, encodes 1020 aminoacids, and has a calculated molecular mass of 108,705 Da. There is aprocaryotic promoter consensus sequence (TATAAT) upstream (at -10) fromthe initiating codon (Doi, R. H., et al., Microbiol. Rev. 50:227-243(1986)). There are no clear homologies in the -35 region assuming aspacing of 5-19 bases upstream from the -10 region (Hawley, D. K., etal., Nucleic Acids Res. 11:2377-2355 (1983)). The probable ribosomalbinding site flanking the 5' end of bca is AGGAGA (Shine, J., et al.,Proc. Natl. Acad. Sci. USA 71:1342-1346 (1974); Gold, L., et al., Annu.Rev. Microbiol. 38:365403 (1981)). Downstream of the TAA terminationcodon are two regions with dyad symmetry that could function astranscription terminators (Brendel, V., et al., Nucleic Acids Res.12:4411-4127 (1984)).

The derived amino acid sequence of the mature peptide of bca predicts aPK_(a) of 4.49, which is close to the experimentally measured values forboth the native and the cloned C protein alpha antigen. The alphaantigen contains no cysteine and only a single methionine at theinitiation codon. The alpha antigen is rich in proline (11% in themature protein) but does not show the XPZ motif identified in the Cprotein beta antigen of GBS (Heden, L. -O., et al., Eur. J. Immunol.21:1481-1490 (1991); Jerlstrom, P. G. et al., Mol. MicrobioL 5:843-849(1991)) or the proline repeat motifs described in M protein of group Astreptococci (Fischetti, V. A., et al., Mol. Microbiol. 4:1603-1605(1990)).

Deduced Signal Sequence of bca and Homologies.

As a cell surface-associated protein, alpha antigen may use a signalsequence to be exported from the cytoplasm. A BLAST search identifiedfive Gram-positive surface proteins with homology to the first 41 aminoacids of the alpha antigen (FIG. 7A). Based on the pattern described forother Gram-positive signal sequences, it is likely that the first 41amino acids of alpha antigen comprise a signal sequence (vonHeijne, G.,Eur. J. Biochem. 133:17-21 (1983); vonHeijne, G. et al., FEBS Lett.244:439-446 (1989)). There is a high proportion of arginine and lysineresidues near the N terminal, followed by a hydrophobic region, a serineat position 36, and a valine at position 41. Other possibilities arecleavages after valine at position 54 or either of the alanine residuesat positions 55 and 56 that follow a serine at position 52. Assumingthat the signal sequence is cleaved following amino acid 41, the matureprotein would contain 979 amino acids with a molecular mass of 104,106Da. This suggests that the signal sequence is encoded by 123nucleotides, making up 4% of the gene, and has a molecular mass of 4616Da. Further support for a signal sequence of this size comes fromWestern blots comparing the sizes of the native and cloned alphaantigens probed with the monoclonal antibody 4G8. As shown in FIG. 8,each of the steps of the alpha antigen protein ladder from clone pJMS23is slightly larger than that of the native protein from GBS A909, whichsuggests that the signal sequence may not be processed in E. coli as itwould be in the GBS. The size difference is about 4 kDa, which wouldcorrespond to a shorter (41 amino acids) rather than a larger (53-55amino acids) signal sequence in bca.

Analysis of the N Terminus of bca.

Following the putative signal sequence, there is a region of 185 aminoacids before the repeated sequences. The N-terminal region contains 555nucleotides, accounts for 18% of the gene, and encodes a polypeptidewith a predicted molecular mass of 20,417 Da. A computer searchcomparing the primary nucleotide sequence and the derived amino acidsequence in all six reading frames of the N terminus of bca withsequences in GenBank and Swiss-Prot using the BLAST network of programsfound no homologies, thus suggesting that this region of the gene isunlike any previously sequenced or described nucleic acid or amino acidsequence.

Repeating Unit Region of bca.

Beginning at amino acid 679 of the DNA sequence, there are nine largetandem repeating units with identical nucleic acid and amino acidstructures that encompass 74% of the gene. The size and repetitivenature of this region of bca are illustrated in FIG. 9. Each repeatingunit consists of 246 nucleotides encoding 82 amino acids with acalculated molecular mass of 8665 Da. The entire repeat region contains749 amino acids and consists of the nine identical repeating units and apartial repeating unit designated 9'. The calculated molecular mass ofthis region is 79,053 Da.

The determination of the beginning and end of the repeat is somewhatarbitrary. Here, the determination starts from the N terminus, beginningwith the first codon that was in the open reading frame. If desired, therepeating units could also be defined as beginning out of frame orstarting at the C-terminal side. BLAST computer searches for nucleicacid and derived amino acid homologies showed to significant matches forthe repeat units. Therefore, these repeating units appear to be uniqueto the alpha antigen and are different in size and structure from thosedescribed for other streptococcal proteins (Heden, L. -O., et al. Eur.J. Immunol. 21:1481-1490 (1991); Jerlstrom, P. G., et al., Mol.Microbiol. 5:843-849 (1991); Fischetti, V. A., et al., in Genetics andMolecular Biology of Streptococci, Lactococci, and Enterococci, Dunny etal. eds. Am. Soc. Microbiol., Washington (1991), pp. 290-294; Yother,J., et al., J. Bacteriol. 174:601-609 (1992)).

C-Terminal Anchor of bca and Homologies.

Following the repeating units is a small C-terminal region containing148 nucleotides and making up 4.4% of the gene. This region encodes 45amino acids with a calculated molecular mass of 4672 Da. A BLAST searchfor amino acid homologies identified a class of Gram-positive surfaceproteins with a common membrane anchor motif (FIG. 7B), including the Mproteins of group A Streptococcus and IgG binding proteins from bothgroup A and group G Streptococcus (Wren, B. W., Mol. Microbiol.5:797-803 (1991)). The amino acid composition at the C terminus ischaracteristic of the peptide membrane anchor, including a hydrophilicstretch with lysine before the LPXTGE SEQ ID NO:2! motif (FIG. 7B)(Fischetti, V. A. et al., Mol. Microbiol. 4:1603-1605 (1990)). This isfollowed by a hydrophobic region with the consensus PPFFXXAA SEQ IDNO:1!, where X designates a hydrophobic amino acid. Finally, there is ahydrophilic tail ending in aspartic acid that presumably extends intothe cytoplasm of the cell.

Analysis of the Nucleotide Sequence and the Deduced Alpha AntigenProtein

FIG. 9 illustrates four distinct regions within the open reading frameof bca as determined from the nucleotide and derived amino acidsequences. A hydrophobicity plot of the amino acid sequence shows thatthe putative signal sequence has a short, hydrophilic N terminus,followed by a hydrophobic stretch, and ending in a hydrophilic region,whereas the C-peptide membrane anchor has a hydrophobic wall-spanningdomain and a small hydrophilic tail (Engelman, D. M., et al., Annu. Rev.Biophys. Biophys. Chem. 15:321-353 (1986); Kyte, J., et al., J. Mol.Biol. 157:105-132 (1982)).

The native alpha antigen demonstrates a ladder of polypeptides atregularly repeating intervals that is also seen with the cloned geneproduct (FIG. 8). The size of the individual repeats in bca could codefor a polypeptide of 8665 Da, which corresponds to the size differencesin the protein ladder. To look at possible mechanisms generating proteinheterogeneity, bca nucleotide and derived RNA and protein sequences weresurveyed. Analysis of the nucleotide sequence of bca failed to showcodons within the repeat regions that could cause early termination oftranslation. In addition, the amino acid sequence of the repeat regionwas screened with the Genetics Computer Group program for potentialsites for proteolytic cleavage. A unique site within each repeat wassensitive to pH 2.5, represented by aspartic acid followed by proline.However, these sites were also found in the N terminus. Although thealpha antigen is relatively resistant to trypsin, there were numerouspotential trypsin cleavage sites found in the sequence. Finally,modeling of RNA sequence and tertiary structure failed to identifyregions within the repeats that might be involved with RNA-mediatedself-cleavage.

Discussion

Two biological properties identified for the alpha antigen of GBS arethe ability to resist opsomophagocytosis in the absence of specificantibody and the expression of epitopes that elicit protectiveantibodies (Madoff, L. C., et al., Infect. Immun. 59:2638-2644 (1991);Lancefield, R. C., et al., J. Exp. Med. 142:165-179 (1975); Payne, N.R., et al., J. Infect. Dis. 151:672-681 (1985); Payne, N. R., et al.,Infect. Immun. 55:1243-1251 (1987)). Analysis of the sequence of thealpha antigen shows four distinct structural domains. The putativeN-terminal signal sequence and the C-terminal membrane anchor supportthe hypothesis that the alpha antigen is a surface-associated membraneprotein. These properties, along with the repeating unit motif, areshared by a number of Gram-positive proteins that are thought to beinvolved in the pathogenesis of bacterial infections (Fischetti, V. A.,et al., in Genetics and Molecular Biology of Streptococci, Lactococci,and Enterococci, Dunny et al. eds. Am. Soc. Microbiol., Washington(1991), pp. 290-294).

The alpha antigen sequence identified a region of large, identical,tandem repeats composing 74% of the gene and demonstrating no homologyto previously described protein or nucleic acids sequences. However, anumber of virulence-associated proteins contain multiple repetitiveelements. The M protein of group A Streptococcus, which isantiphagocytic, carries protective epitopes and displays variability inantigen size and presentation, contains two extended tandem repeatregions and one nontandem repeat region occupying nearly two-thirds ofthe gene (Fischetti, V. A., et al., Rev. Infect. Dis. S356-S359 (1988);Hollingshead, S. K., et al., J. Biol. Chem. 261:1677-1686 (1986);Haanes, E. J., et al., J. Bacteriol. 171:6397-6408 (1989)). Theindividual repeats are smaller in M protein than in the alpha antigenand range from 21 to 81 base pairs. In addition, there is divergencebetween the repeating units at the ends of the repeat region, whilethose in the middle are nearly identical. Pneumococcal surface protein Acontains a region containing up to 10 repetitive segments of 20 aminoacids each (Yother, J. et al., J. Bacteriol. 174:601-609 (1992)). Both Mprotein and pneumococcal surface protein A demonstrate antigenicvariability and changes in protein/gene size thought to be mediated byrepetitive DNA sequences in their structural genes (Fischetti, V. A., etal., in Genetics and Molecular Biology of Streptococci, Lactococci, andEnterococci, Dunny et al. eds. Am. Soc. Microbiol., Washington (1991),pp. 290-294; Yother, J., et al., J. Bacteriol. 174:601-609 (1992);Haanes, E. J., et al., J. Bacteriol. 171:6397-6408 (1989)). OtherGram-positive genes with repetitive motifs include the glycotransferasegenes from Streptococcus sobrinus and Streptococcus mutans (Ferretti, J.J., et al., J. Bacteriol. 169:42714278 (1987); Shiroza, T., et al., J.Bacteriol. 170.810-816 (1988)). Immunodominant epitopes associated withrepetitive sequences have been identified in a number of other pathogensincluding Rickettsia rickettsii, Trichomonas vaginalis, and Clostridiumdifficile (Dailey, D. C., et al., Infect. Immun. 59:2083-2088 (1991);Anderson, B. E., et al., Infect. Immun. 58:2760-2769 (1990);vonEichel-Streiber, C., et al., Gene 96:107-113 (1990)). The repeatsfound in alpha antigens are unique for three reasons: (i) They arelarger than those found for other Gram-positive surface proteins. (ii)They are identical at the nucleic acid level and do not diverge. (iii)The size of protein encoded by the repeating units corresponds to theladdering seen in the native and cloned alpha antigens.

The findings of large tandem repeating units raises many questions aboutthe genotypic and phenotypic variability of the alpha antigen. Whenprobed on Western immunoblots with the 4G8 monoclonal antibody, both thenative and the cloned alpha antigen display a regular ladder of proteinsvarying by about 8 kDa, and the size of the alpha antigen varies betweenstrains (Madoff, L. C., et al. Infect. Immun. 59:2638-2644 (1991)).Restriction endonuclease mapping of the original alpha antigen clonepJMS23 showed multiple Sty I fragments of about 270 base pairs (Michel,J. L., et al., Infect. Immun. 59:2023-2028 (1991)). Since strain A909contains only one copy of bca it was proposed that these fragments maybe responsible for the protein heterogeneity. The nucleotide sequenceconfirms the repetitive nature of the gene but does not identify themechanism of protein laddering.

Since multiple protein sizes are seen in both native and clonedbackgrounds and since there is no evidence for a gene family, wepostulate that laddering results from a mechanism common to both E. coliand GBS and/or is mediated by a property specific to the alpha antigen.Western blots on Tn5 transposon insertion mutations within the repeatregion still show laddering, which demonstrates that the C terminus isnot required for heterogeneity, suggesting that either the N-terminal orrepeat region determines laddering.

Studies of the alpha antigen among GBS isolates using a monoclonalantibody showed that the maximum molecular size of the alpha antigen isconstant for a given isolate but varies widely among different isolates(Madoff, L. C., et al., Infect. Immun. 59:2638-2644 (1991)). The tandemrepeating units could provide convenient fixed recombination sites fordeletion or duplication of the repeat region. Deletion would reduce thesize of the gene and might occur during DNA replication by unequalcrossover or mispaired template slippage, which would occur in frame(Harayama, S., et al., J. Bacteriol 173:7540-7548 (1991)). Duplicationof DNA could be a mechanism to amplify mutations within a repeat andcreate antigenic diversity. However, we have no evidence that thevariation in the protein size of the alpha antigen is accompanied byantigenic diversity and the expression of different protective oropsonic epitopes.

The nine complete tandem repeats in the alpha antigen from A909 areidentical at the nucleic acid level, which demonstrates a highlyconserved structure. This suggests that the duplication causing therepeats is a recent event, that there are properties internal to therepeats that maintain their integrity, or that their structure isessential for the gene. Southern blots of genomic DNA from alphaantigen-bearing strains of GBS probed with alpha antigen-specific DNAshow variability in gene size among strains. To look at the mechanism ofgenotypic diversity among strains, it will be necessary to clone andsequence bca from other phenotypic variants and to determine thephylogenetic relationships among C protein-bearing strains of GBS(Michel, J. L., et al. in Genetics and Molecular Biology ofStreptococci, Lactococci, and Enterococd, Dunny, G. M., et al. eds., Am.Soc. Microbiol., Washington (1991), pp. 214-218; Michel, J. L., et al.,Infect. Immun. 59:2023-2028 (1991); Cleat, P. H., et al., Infect. Immun.55:1151-1155 (1987); Heden, L. -O., et al. Eur. J. Immunol. 21:1481-1490(1991); Lindahl, G., et al., Eur. J. Immunol. 20:2241-2247 (1990)).

Therefore, in summary, Western blots of both the native alpha antigenand the cloned gene product demonstrate a regularly laddered pattern ofheterogeneous polypeptides. The nucleotide sequence of the bca locusreveals an open reading frame of 3060 nucleotides encoding a precursorprotein of 108,705 Da. Cleavage of a putative signal sequence of 41amino acids yields a mature protein of 104,106 Da. The 20,417-DaN-terminal region of the alpha antigen shows no homology to previouslydescribed protein sequences and is followed by a series of nine tandemrepeating units that make up 74% of the mature protein. Each repeatingunit is identical and consists of 82 amino acids with a molecular massof 8665 Da, which is encoded by 246 nucleotides. The size of therepeating units corresponds to the observed size differences in theheterogeneous ladder of alpha C proteins expressed by GBS. TheC-terminal region of the alpha antigen contains a membrane anchor domainmotif that is shared by a number of Gram-positive surface proteins. Thelarge region of identical repeating units in bca defines protectiveepitopes and its structure may be manipulated for the construction ofprotective vaccines that are directed to the phenotypic and genotypicdiversity of the alpha antigen.

EXAMPLE 15 A Vaccine Containing C Protein Alpha Antigen FunctionalDerivatives Having at Least one of the Native Repeating Units

The above-described protective C protein alpha antigen functionalderivatives (such as a protein moiety of N, C, N--C, R₁, R₂, R₃, R₄, R₅,R₆, R₇, R₈, R₉, N--R₁, N--R₂, N--R₃, N--R₄, N--R₅, N--R₆, N--R₇, N--R₈,N--R₉, R₁ --C, R₂ --C, R₃ --C, R₄ --C, R₅ --C, R₆ --C, R₇ --C, R₈ --C,R₉ --C, N--R₁ --C, N--R₂ --C, N--R₃ --C, N--R₄ --C, N--R₅ --C, N--R₆--C, N--R₇ --C, N--R₈ --C, or N--R₉ --C) may be prepared by recombinantmeans using recombinant methods similar to those described above forcloning and expressing the native group B Streptococcus alpha antigenand beta antigen in hosts such as E. coli. Any technique may be utilizedto synthesize the desired alpha antigen functional derivative sequence,including those described above for the recombinant production of theseproteins, and those described by Williams, J. I., et al., U.S. Pat, No.5,089,406 ("Method of Producing a Gene Cassette Coding for Polypeptideswith Repeating Amino Acid Sequences," incorporated herein by reference)and by McPherson, M. J., ed., Directed Mutagenesis, A PracticalApproach," IRL Press, New York, 1991.

The recombinantly expressed, above-described protective C protein alphaantigen functional derivatives (such as a protein moiety of N, C, N--C,R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, N--R₁, N--R₂, N--R₃, N--R₄, N--R₅,N--R₆, N--R₇, N--R₈, N--R₉, R₁ --C, R₂ --C, R₃ --C, R₄ --C, R₅ --C, R₆--C, R₇ --C, R₈ --C, R₉ --C, N--R₁ --C, N--R₂ --C, N--R₃ --C, N--R₄ --C,N--R₅ --C, N--R₆ --C, N--R₇ --C, N--R₈ --C, or N--R₉ --C) may bepurified, if necessary, from the recombinant host or medium usingtechniques known in the art and then tested for their potential in aconjugate vaccine. Each peptide species may be tested alone, or incombination with other peptides. To assess this potential, cellularextracts of E. coli containing recombinant plasmids are prepared asdescribed above, and used to immunize rabbits. The resulting antiseraare tested in the mouse lethality model for their ability to protectmice from infection by the group B Streptococcus strain H36B. StrainH36B carries the C protein of group B Streptococcus. As a control, theability of the antisera to protect the mice against infection byStreptococcus strain 515 (which does not carry the C protein) isdetermined.

A similar assay may be used to assess the conjugated form wherein thepeptide is conjugated to a group B Streptococcus polysaccharide usingthe above described techniques known in the art. Preferably, this is agroup B Streptococcus capsid polysaccharide. The conjugates are used toimmunize rabbits. The resulting antisera are tested in the mouselethality model for their ability to protect mice from infection by thegroup B Streptococcus strain H36B. Strain H36B carries the C protein ofgroup B Streptococcus. As a control, the ability of the antisera toprotect the mice against infection by Streptococcus strain 515 (whichdoes not carry the C protein) is determined.

Although the foregoing refers to particular preferred embodiments, itwill be understood that the present invention is not so limited. It willoccur to those ordinarily skilled in the art that various modificationsmay be made to the disclosed embodiments and that such modifications areintended to be within the scope of the present invention.

    __________________________________________________________________________    #             SEQUENCE LISTING    - (1) GENERAL INFORMATION:    -    (iii) NUMBER OF SEQUENCES:  65    - (2) INFORMATION FOR SEQ ID NO:1:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 8 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:    -      Pro Pro Phe Phe Xaa Xaa Ala Ala    #  5 1    - (2) INFORMATION FOR SEQ ID NO:2:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 6 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:    -      Leu Pro Xaa Thr Gly Glu    #  5 1    - (2) INFORMATION FOR SEQ ID NO:3:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 15 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: both    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:    #    15    - (2) INFORMATION FOR SEQ ID NO:4:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 11 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: both    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:    #       11    - (2) INFORMATION FOR SEQ ID NO:5:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 12 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: both    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:    #       12    - (2) INFORMATION FOR SEQ ID NO:6:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 57 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: both    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:    - ATGACCATGA TTACGAATTC GAGCTCGCCC GGGGATCCAT TGTGCTGGAA AG - #CCACC      57    - (2) INFORMATION FOR SEQ ID NO:7:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 16 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: both    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:    #    16    - (2) INFORMATION FOR SEQ ID NO:8:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 32 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: both    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:    #          32      CCAG CACAATGGAT CC    - (2) INFORMATION FOR SEQ ID NO:9:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 12 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: both    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:    #       12    - (2) INFORMATION FOR SEQ ID NO:10:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: both    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:    # 20               AAAG    - (2) INFORMATION FOR SEQ ID NO:11:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 12 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: both    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:    #       12    - (2) INFORMATION FOR SEQ ID NO:12:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 13 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: both    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:    #      13    - (2) INFORMATION FOR SEQ ID NO:13:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 15 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: double              (D) TOPOLOGY: both    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:    #    15    - (2) INFORMATION FOR SEQ ID NO:14:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 1380 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ix) FEATURE:              (A) NAME/KEY: CDS              (B) LOCATION: 79..1173    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 1004    #/note= "This feature is to signify    #nucleotide sequence from position 757                   through 1 - #003 is inserted at position 1004 and can be    #up to eight times (for a total of nine    #copies of these sequences within the                   polynucleoti - #de)."    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:    - AGCATAGATA TTCTAATATT TGTTGTTTAA GCCTATAATT TACTCTGTAT AG - #AGTTATAC      60    #AAC AGT TAT GAT      111 TTT AGA AGG TCT AAA AAT    #Tyr Asphe Arg Arg Ser Lys Asn Asn Ser    # 10    - ACT TCA CAG ACG AAA CAA CGG TTT TCA ATT AA - #G AAG TTC AAG TTT GGT     159    Thr Ser Gln Thr Lys Gln Arg Phe Ser Ile Ly - #s Lys Phe Lys Phe Gly    #             25    - GCA GCT TCT GTA CTA ATT GGT CTT AGT TTT TT - #G GGT GGG GTT ACA CAA     207    Ala Ala Ser Val Leu Ile Gly Leu Ser Phe Le - #u Gly Gly Val Thr Gln    #         40    - GGT AAT CTT AAT ATT TTT GAA GAG TCA ATA GT - #T GCT GCA TCT ACA ATT     255    Gly Asn Leu Asn Ile Phe Glu Glu Ser Ile Va - #l Ala Ala Ser Thr Ile    #     55    - CCA GGG AGT GCA GCG ACC TTA AAT ACA AGC AT - #C ACT AAA AAT ATA CAA     303    Pro Gly Ser Ala Ala Thr Leu Asn Thr Ser Il - #e Thr Lys Asn Ile Gln    # 75    - AAC GGA AAT GCT TAC ATA GAT TTA TAT GAT GT - #A AAA TTA GGT AAA ATA     351    Asn Gly Asn Ala Tyr Ile Asp Leu Tyr Asp Va - #l Lys Leu Gly Lys Ile    #                 90    - GAT CCA TTA CAA TTA ATT GTT TTA GAA CAA GG - #T TTT ACA GCA AAG TAT     399    Asp Pro Leu Gln Leu Ile Val Leu Glu Gln Gl - #y Phe Thr Ala Lys Tyr    #            105    - GTT TTT AGA CAA GGT ACT AAA TAC TAT GGG GA - #T GTT TCT CAG TTG CAG     447    Val Phe Arg Gln Gly Thr Lys Tyr Tyr Gly As - #p Val Ser Gln Leu Gln    #       120    - AGT ACA GGA AGG GCT AGT CTT ACC TAT AAT AT - #A TTT GGT GAA GAT GGA     495    Ser Thr Gly Arg Ala Ser Leu Thr Tyr Asn Il - #e Phe Gly Glu Asp Gly    #   135    - CTA CCA CAT GTA AAG ACT GAT GGA CAA ATT GA - #T ATA GTT AGT GTT GCT     543    Leu Pro His Val Lys Thr Asp Gly Gln Ile As - #p Ile Val Ser Val Ala    140                 1 - #45                 1 - #50                 1 -    #55    - TTA ACT ATT TAT GAT TCA ACA ACC TTG AGG GA - #T AAG ATT GAA GAA GTT     591    Leu Thr Ile Tyr Asp Ser Thr Thr Leu Arg As - #p Lys Ile Glu Glu Val    #               170    - AGA ACG AAT GCA AAC GAT CCT AAG TGG ACG GA - #A GAA AGT CGT ACT GAG     639    Arg Thr Asn Ala Asn Asp Pro Lys Trp Thr Gl - #u Glu Ser Arg Thr Glu    #           185    - GTT TTA ACA GGA TTA GAT ACA ATT AAG ACA GA - #T ATT GAT AAT AAT CCT     687    Val Leu Thr Gly Leu Asp Thr Ile Lys Thr As - #p Ile Asp Asn Asn Pro    #       200    - AAG ACG CAA ACA GAT ATT GAT AGT AAA ATT GT - #T GAG GTT AAT GAA TTA     735    Lys Thr Gln Thr Asp Ile Asp Ser Lys Ile Va - #l Glu Val Asn Glu Leu    #   215    - GAG AAA TTG TTA GTA TTG TCA GTA CCG GAT AA - #A GAT AAA TAT GAT CCA     783    Glu Lys Leu Leu Val Leu Ser Val Pro Asp Ly - #s Asp Lys Tyr Asp Pro    220                 2 - #25                 2 - #30                 2 -    #35    - ACA GGA GGG GAA ACA ACA GTA CCC CAA GGG AC - #A CCA GTT TCA GAT AAA     831    Thr Gly Gly Glu Thr Thr Val Pro Gln Gly Th - #r Pro Val Ser Asp Lys    #               250    - GAA ATC ACA GAC TTA GTT AAG ATT CCA GAT GG - #C TCA AAA GGG GTT CCG     879    Glu Ile Thr Asp Leu Val Lys Ile Pro Asp Gl - #y Ser Lys Gly Val Pro    #           265    - ACA GTT GTT GGT GAT CGT CCA GAT ACT AAC GT - #T CCT GGA GAT CAT AAA     927    Thr Val Val Gly Asp Arg Pro Asp Thr Asn Va - #l Pro Gly Asp His Lys    #       280    - GTA ACG GTA GAA GTA ACG TAT CCA GAT GGA AC - #A AAG GAT ACA GTA GAA     975    Val Thr Val Glu Val Thr Tyr Pro Asp Gly Th - #r Lys Asp Thr Val Glu    #   295    - GTA ACG GTT CAT GTG ACA CCA AAA CCA GTA CC - #G GAT AAA GAT AAA TAT    1023    Val Thr Val His Val Thr Pro Lys Pro Val Pr - #o Asp Lys Asp Lys Tyr    300                 3 - #05                 3 - #10                 3 -    #15    - GAT CCA ACA GGT AAA GCT CAG CAA GTC AAC GG - #T AAA GGA AAT AAA CTA    1071    Asp Pro Thr Gly Lys Ala Gln Gln Val Asn Gl - #y Lys Gly Asn Lys Leu    #               330    - CCA GCA ACA GGT GAG AAT GCA ACT CCA TTC TT - #T AAT GTT GCA GCT TTG    1119    Pro Ala Thr Gly Glu Asn Ala Thr Pro Phe Ph - #e Asn Val Ala Ala Leu    #           345    - ACA ATT ATA TCA TCA GTT GGT TTA TTA TCT GT - #T TCT AAG AAA AAA GAG    1167    Thr Ile Ile Ser Ser Val Gly Leu Leu Ser Va - #l Ser Lys Lys Lys Glu    #       360    - GAT TAATCTTTTG ACCTAAAATG TCACTAAATT TTTCACCATT TATTGGTGT - #G    1220    Asp        365    - AACACATTAA TAAAGTTATG CATCTCTCTC CAACAAAATT AATTAAAGTG TT - #TCAATTTT    1280    - TCGAGATTAA TTCTTGAAAA AAGCCTATCG AGATTATTAA TTTCGATAGG CT - #TTTGATTT    1340    #  1380            ATAC CTTGTTATTG GACGCTTACT    - (2) INFORMATION FOR SEQ ID NO:15:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 364 amino              (B) TYPE: amino acid              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: protein    -     (ix) FEATURE:              (A) NAME/KEY: misc.sub.-- - #feature              (B) LOCATION: 310    #/note= "This feature indicates that                   the amino - # acid sequence from position 227 through    #inserted at position 310 and may repeat up to                   eight tim - #es (for a total of nine repeating copies of                   these seq - #uences within the polypeptide)."    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:    - Met Phe Arg Arg Ser Lys Asn Asn Ser Tyr As - #p Thr Ser Gln Thr Lys    #                 15    - Gln Arg Phe Ser Ile Lys Lys Phe Lys Phe Gl - #y Ala Ala Ser Val Leu    #             30    - Ile Gly Leu Ser Phe Leu Gly Gly Val Thr Gl - #n Gly Asn Leu Asn Ile    #         45    - Phe Glu Glu Ser Ile Val Ala Ala Ser Thr Il - #e Pro Gly Ser Ala Ala    #     60    - Thr Leu Asn Thr Ser Ile Thr Lys Asn Ile Gl - #n Asn Gly Asn Ala Tyr    # 80    - Ile Asp Leu Tyr Asp Val Lys Leu Gly Lys Il - #e Asp Pro Leu Gln Leu    #                 95    - Ile Val Leu Glu Gln Gly Phe Thr Ala Lys Ty - #r Val Phe Arg Gln Gly    #           110    - Thr Lys Tyr Tyr Gly Asp Val Ser Gln Leu Gl - #n Ser Thr Gly Arg Ala    #       125    - Ser Leu Thr Tyr Asn Ile Phe Gly Glu Asp Gl - #y Leu Pro His Val Lys    #   140    - Thr Asp Gly Gln Ile Asp Ile Val Ser Val Al - #a Leu Thr Ile Tyr Asp    145                 1 - #50                 1 - #55                 1 -    #60    - Ser Thr Thr Leu Arg Asp Lys Ile Glu Glu Va - #l Arg Thr Asn Ala Asn    #               175    - Asp Pro Lys Trp Thr Glu Glu Ser Arg Thr Gl - #u Val Leu Thr Gly Leu    #           190    - Asp Thr Ile Lys Thr Asp Ile Asp Asn Asn Pr - #o Lys Thr Gln Thr Asp    #       205    - Ile Asp Ser Lys Ile Val Glu Val Asn Glu Le - #u Glu Lys Leu Leu Val    #   220    - Leu Ser Val Pro Asp Lys Asp Lys Tyr Asp Pr - #o Thr Gly Gly Glu Thr    225                 2 - #30                 2 - #35                 2 -    #40    - Thr Val Pro Gln Gly Thr Pro Val Ser Asp Ly - #s Glu Ile Thr Asp Leu    #               255    - Val Lys Ile Pro Asp Gly Ser Lys Gly Val Pr - #o Thr Val Val Gly Asp    #           270    - Arg Pro Asp Thr Asn Val Pro Gly Asp His Ly - #s Val Thr Val Glu Val    #       285    - Thr Tyr Pro Asp Gly Thr Lys Asp Thr Val Gl - #u Val Thr Val His Val    #   300    - Thr Pro Lys Pro Val Pro Asp Lys Asp Lys Ty - #r Asp Pro Thr Gly Lys    305                 3 - #10                 3 - #15                 3 -    #20    - Ala Gln Gln Val Asn Gly Lys Gly Asn Lys Le - #u Pro Ala Thr Gly Glu    #               335    - Asn Ala Thr Pro Phe Phe Asn Val Ala Ala Le - #u Thr Ile Ile Ser Ser    #           350    - Val Gly Leu Leu Ser Val Ser Lys Lys Lys Gl - #u Asp    #       360    - (2) INFORMATION FOR SEQ ID NO:16:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 56 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:    - Met Phe Arg Arg Ser Lys Asn Asn Ser Tyr As - #p Thr Ser Gln Thr Lys    #                15    - Gln Arg Phe Ser Ile Lys Lys Phe Lys Phe Gl - #y Ala Ala Ser Val Leu    #            30    - Ile Gly Leu Ser Phe Leu Gly Gly Val Thr Gl - #n Gly Asn Leu Asn Ile    #        45    - Phe Glu Glu Ser Ile Val Ala Ala    #    55    - (2) INFORMATION FOR SEQ ID NO:17:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 37 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:    - Met Phe Lys Ser Asn Tyr Glu Arg Lys Met Ar - #g Tyr Ser Ile Arg Lys    #                15    - Phe Ser Val Gly Val Ala Ser Val Ala Val Ar - #g Ser Leu Phe Met Gly    #            30    - Ser Val Ala His Ala            35    - (2) INFORMATION FOR SEQ ID NO:18:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 41 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:    - Met Ala Arg Gln Gln Thr Lys Lys Asn Tyr Se - #r Leu Arg Lys Leu Lys    #                15    - Thr Gly Thr Ala Ser Val Ala Val Ala Leu Th - #r Val Leu Gly Ala Gly    #            30    - Phe Ala Asn Gln Thr Glu Val Arg Ala    #        40    - (2) INFORMATION FOR SEQ ID NO:19:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 42 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:19:    - Met Thr Lys Asn Asn Thr Asn Arg His Tyr Se - #r Leu Arg Lys Leu Lys    #                15    - Thr Gly Thr Ala Ser Val Ala Val Ala Leu Th - #r Val Leu Gly Ala Gly    #            30    - Leu Val Val Asn Thr Asn Glu Val Ser Ala    #        40    - (2) INFORMATION FOR SEQ ID NO:20:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 41 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:20:    - Met Ala Lys Asn Asn Thr Asn Arg His Tyr Se - #r Leu Arg Lys Leu Lys    #                15    - Thr Gly Thr Ala Ser Val Ala Val Ala Leu Th - #r Val Leu Gly Ala Gly    #            30    - Phe Ala Asn Gln Thr Glu Val Lys Ala    #        40    - (2) INFORMATION FOR SEQ ID NO:21:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 50 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:21:    - Met Ala Lys Asn Asn Thr Asn Arg His Tyr Se - #r Leu Arg Lys Leu Lys    #                15    - Thr Gly Thr Ala Ser Val Ala Val Ala Leu Th - #r Val Leu Gly Ala Gly    #            30    - Phe Ala Asn Gln Thr Glu Val Lys Ala Asn Gl - #y Asp Gly Asn Pro Arg    #        45    - Glu Val        50    - (2) INFORMATION FOR SEQ ID NO:22:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 45 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:22:    - Lys Ala Gln Gln Val Asn Gly Lys Gly Asn Ly - #s Leu Pro Ala Thr Gly    #                15    - Glu Asn Ala Thr Pro Phe Phe Asn Val Ala Al - #a Leu Thr Ile Ile Ser    #            30    - Ser Val Gly Leu Leu Ser Val Ser Lys Lys Ly - #s Glu Asp    #        45    - (2) INFORMATION FOR SEQ ID NO:23:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 46 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:23:    - Asn Lys Ala Pro Met Lys Glu Thr Lys Arg Gl - #n Leu Pro Tyr Thr Gly    #                15    - Val Thr Ala Asn Pro Phe Phe Thr Ala Ala Al - #a Leu Thr Val Met Ala    #            30    - Thr Ala Gly Val Ala Ala Val Val Lys Arg Ly - #s Glu Glu Asn    #        45    - (2) INFORMATION FOR SEQ ID NO:24:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 45 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:24:    - Arg Pro Ser Gln Asn Lys Gly Met Arg Ser Gl - #n Leu Pro Ser Thr Gly    #                15    - Glu Ala Ala Asn Pro Phe Phe Thr Ala Ala Al - #a Ala Thr Val Met Val    #            30    - Ser Ala Gly Met Leu Ala Leu Lys Arg Lys Gl - #u Glu Asn    #        45    - (2) INFORMATION FOR SEQ ID NO:25:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 46 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:25:    - Ala Lys Lys Glu Asp Ala Lys Lys Ala Glu Th - #r Leu Pro Thr Thr Gly    #                15    - Glu Gly Ser Asn Pro Phe Phe Thr Ala Ala Al - #a Leu Ala Val Met Ala    #            30    - Gly Ala Gly Ala Leu Ala Val Ala Ser Lys Ar - #g Lys Glu Asp    #        45    - (2) INFORMATION FOR SEQ ID NO:26:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 46 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:26:    - Ala Lys Lys Asp Asp Ala Lys Lys Ala Glu Th - #r Leu Pro Thr Thr Gly    #                15    - Glu Gly Ser Asn Pro Phe Phe Thr Ala Ala Al - #a Leu Ala Val Met Ala    #            30    - Gly Ala Gly Ala Leu Ala Val Ala Ser Lys Ar - #g Lys Glu Asp    #        45    - (2) INFORMATION FOR SEQ ID NO:27:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 45 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:27:    - Ser Arg Ser Ala Met Thr Gln Gln Lys Arg Th - #r Leu Pro Ser Thr Gly    #                15    - Glu Thr Ala Asn Pro Phe Phe Thr Ala Ala Al - #a Ala Thr Val Met Val    #            30    - Ser Ala Gly Met Leu Ala Leu Lys Arg Lys Gl - #u Glu Asn    #        45    - (2) INFORMATION FOR SEQ ID NO:28:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 34 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:28:    - Asn Lys Ala Pro Met Lys Glu Thr Lys Arg Gl - #n Leu Pro Ser Thr Gly    #                15    - Glu Thr Ala Asn Pro Phe Phe Thr Ala Ala Al - #a Leu Thr Val Met Ala    #            30    - Ala Ala    - (2) INFORMATION FOR SEQ ID NO:29:    -      (i) SEQUENCE CHARACTERISTICS:    #acids    (A) LENGTH: 44 amino              (B) TYPE: amino acid              (D) TOPOLOGY: both    -     (ii) MOLECULE TYPE: peptide    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:29:    - Lys Gly Asn Pro Thr Ser Thr Thr Glu Lys Ly - #s Leu Pro Tyr Thr Gly    #                15    - Val Ala Ser Asn Leu Val Leu Glu Ile Met Gl - #y Leu Leu Gly Leu Ile    #            30    - Gly Thr Ser Phe Ile Ala Met Lys Arg Arg Ly - #s Ser    #        40    - (2) INFORMATION FOR SEQ ID NO:30:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 5 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:30:    #             5    - (2) INFORMATION FOR SEQ ID NO:31:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 5 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:31:    #             5    - (2) INFORMATION FOR SEQ ID NO:32:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 15 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:32:    #    15    - (2) INFORMATION FOR SEQ ID NO:33:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 11 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:33:    #       11    - (2) INFORMATION FOR SEQ ID NO:34:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 16 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:34:    #    16    - (2) INFORMATION FOR SEQ ID NO:35:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 16 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:35:    #    16    - (2) INFORMATION FOR SEQ ID NO:36:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 16 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:36:    #    16    - (2) INFORMATION FOR SEQ ID NO:37:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 32 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:37:    #          32      GGTC GTGTTACCTA GG    - (2) INFORMATION FOR SEQ ID NO:38:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 8 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:38:    #           8    - (2) INFORMATION FOR SEQ ID NO:39:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 8 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:39:    #           8    - (2) INFORMATION FOR SEQ ID NO:40:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 12 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:40:    #       12    - (2) INFORMATION FOR SEQ ID NO:41:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 12 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:41:    #       12    - (2) INFORMATION FOR SEQ ID NO:42:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 8 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:42:    #           8    - (2) INFORMATION FOR SEQ ID NO:43:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:43:    # 20               ATCC    - (2) INFORMATION FOR SEQ ID NO:44:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:44:    # 20               TAGG    - (2) INFORMATION FOR SEQ ID NO:45:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 8 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:45:    #           8    - (2) INFORMATION FOR SEQ ID NO:46:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 12 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:46:    #       12    - (2) INFORMATION FOR SEQ ID NO:47:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 20 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:47:    # 20               TTTC    - (2) INFORMATION FOR SEQ ID NO:48:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 6 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:48:    #            6    - (2) INFORMATION FOR SEQ ID NO:49:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 7 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:49:    #           7    - (2) INFORMATION FOR SEQ ID NO:50:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 12 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:50:    #       12    - (2) INFORMATION FOR SEQ ID NO:51:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 10 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:51:    #        10    - (2) INFORMATION FOR SEQ ID NO:52:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 9 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:52:    #          9    - (2) INFORMATION FOR SEQ ID NO:53:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 6 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:53:    #            6    - (2) INFORMATION FOR SEQ ID NO:54:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 13 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:54:    #      13    - (2) INFORMATION FOR SEQ ID NO:55:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 15 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:55:    #    15    - (2) INFORMATION FOR SEQ ID NO:56:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 10 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:56:    #        10    - (2) INFORMATION FOR SEQ ID NO:57:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 9 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:57:    #          9    - (2) INFORMATION FOR SEQ ID NO:58:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 6 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:58:    #            6    - (2) INFORMATION FOR SEQ ID NO:59:    -      (i) SEQUENCE CHARACTERISTICS:    #pairs    (A) LENGTH: 13 base              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:59:    #      13    - (2) INFORMATION FOR SEQ ID NO:60:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 5 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:60:    #             5    - (2) INFORMATION FOR SEQ ID NO:61:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 5 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:61:    #             5    - (2) INFORMATION FOR SEQ ID NO:62:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 4 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:62:    #              4    - (2) INFORMATION FOR SEQ ID NO:63:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 4 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:63:    #              4    - (2) INFORMATION FOR SEQ ID NO:64:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 9 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:64:    #          9    - (2) INFORMATION FOR SEQ ID NO:65:    -      (i) SEQUENCE CHARACTERISTICS:              (A) LENGTH: 9 base p - #airs              (B) TYPE: nucleic acid              (C) STRANDEDNESS: single              (D) TOPOLOGY: linear    -     (ii) MOLECULE TYPE: cDNA    -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:65:    #          9    __________________________________________________________________________

What is claimed is:
 1. A conjugate vaccine that confers host immunity toan infection in a mammal by group B Streptococcus which comprises (a) agroup-specific or type-specific group B Streptococcus polysaccharideconjugated to (b) a functional derivative of the group B Streptococcus Cprotein alpha antigen, wherein said derivative elicits protectiveantibodies against said group B Streptococcus.
 2. A method forpreventing or attenuating an infection in a mammal caused by a group BStreptococcus that comprises administering to an individual, aneffective amount of a conjugate vaccine, said conjugate vaccineconferring protective host immunity against an infection by group BStreptococcus wherein said vaccine comprises a therapeutically effectiveamount of:(a) a capsular polysaccharide that elicits antibodies to groupB Streptococcus; conjugated to (b) a C protein alpha antigen or betaantigen of said group B Streptococcus, wherein said alpha antigen is agroup B Streptococcus protein of at least 40,000 daltons that isrecognized by antiserum to the C protein encoded by pJMS23, and whereinsaid beta antigen is a group B Streptococcus protein of at least 50,000daltons that is recognized by antiserum to the protein encoded bypJMS1;wherein said vaccine is substantially free of streptococcalproteins other than said C protein alpha antigen or said C protein betaantigen, said conjugate vaccine is in a pharmacologically acceptablecomposition and wherein both said capsular polysaccharide and said Cprotein contributes to the development of said protective host immunityto said infection.
 3. The method of claim 2, wherein said C protein ofthe conjugate vaccine is encoded by a plasmid selected from the groupconsisting of pJMS23 and plasmid pJMS1.
 4. The method of claim 2,wherein said C protein of the conjugate vaccine is said alpha antigen.5. The method of claim 4, wherein said alpha antigen is that encoded byplasmid pJMS23.
 6. The method of claim 2, wherein said C protein of theconjugate vaccine is said beta antigen.
 7. The method of claim 6,wherein said beta antigen is that encoded by plasmid pJMS1.
 8. A methodfor preventing or attenuating an infection in a mammal caused by a groupB Streptococcus that comprises administering to an individual, aneffective amount of a conjugate vaccine, said conjugate vaccineconferring protective host immunity against an infection by group BStreptococcus wherein said vaccine comprises a therapeutically effectiveamount of: (a) a group B Streptococcus capsular polysaccharide thatelicits antibodies to group B Streptococcus; conjugated to (b) afunctional derivative selected from the group B Streptococcus C proteinsconsisting of an alpha antigen, a beta antigen, fragments of said alphaantigen, fragments of said beta antigen and combinations thereof,wherein said derivative elicits protective antibodies against said groupB Streptococcus.
 9. The method of claim 8, wherein said polysaccharideof the conjugate vaccine is a capsular polysaccharide.
 10. The method ofclaim 8, wherein said C protein alpha antigen derivative of saidconjugate vaccine is selected from the group consisting of N, C, N--C,R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R_(9'), R_(x), N--R₁, N--R₂, N--R₃,N--R₄, N--R₅, N--R₆, N--R₇, N--R₈, N--R₉, N--R_(9'), N--R_(x), R₁ --C,R₂ --C, R₃ --C, R₄ --C, R₅ --C, R₆ --C, R₇ --C, R₈ --C, R₉ --C, R_(9')--C, R_(x) --C, N--R₁ --C, N--R₂ --C, N--R₃ --C, N--R₄ --C, N--R₅ --C,N--R₆ --C, N--R₇ --C, N--R₈ --C, N--R₉ --C, N--R_(9') --C; N--R_(x) --Cand combinations thereof where "X" is 10 or greater, where "N" is theN-terminal sequence that precedes the start of the alpha antigenrepeating unit sequence that is found in the sequence shown in FIG. 6(SEQ ID NO:15), with or without the signal sequence, "C" is the 45 aminoacid C-terminal anchor sequence as shown on FIG. 6 (SEQ ID NO:15), "R"is one copy of the 82 amino acids 227-308 of the sequence of FIG. 6 (SEQID NO:15), and "R_(x) " is "X" number of tandem copies of this repeat,tandemly joined at the carboxyl end of one R unit to the amino terminalend of the adjoining R unit, and 9' represents the sequence of aminoacids 227-975 as shown in FIG. 6 (SEQ ID NO:15).
 11. The method of claim8, wherein said derivative is selected from the group consisting of N,C, R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R_(9'), R_(x) where "X" is 10 orgreater, and combinations thereof.
 12. The method of claim 11, whereinsaid derivative is selected from the group consisting of N, C, R₁, R₂,R₃, R₄, R₅, R₆, R₇, R₈, R₉, R_(9'), and R_(x) where "X" is 10 orgreater.
 13. The method of claim 10, wherein said derivative is selectedfrom the group consisting of N--C, N--R₁, N--R₂, N--R₃, N--R₄, N--R₅,N--R₆, N--R₇, N--R₈, N--R₉, N--R_(9'), N--R_(x) where "X" is 10 orgreater, and combinations thereof.
 14. The method of claim 13, whereinsaid derivative is selected from the group consisting of N--C, N--R₁,N--R₂, N--R₃, N--R₄, N--R₅, N--R₆, N--R₇, N--R₈, N--R₉, N--R_(9'), andN--R_(x) where "X" is 10 or greater.
 15. The method of claim 10, whereinsaid derivative is selected from the group consisting of R₁ --C, R₂ --C,R₃ --C, R₄ --C, R₅ --C, R₆ --C, R₇ --C, R₈ --C, R₉ --C, R_(9') --C,R_(x) --C where "X" is 10 or greater, and combinations thereof.
 16. Themethod of claim 15 wherein said derivative is selected from the groupconsisting of R₁ --C, R₂ --C, R₃ --C, R₄ --C, R₅ --C, R₆ --C, R₇ --C, R₈--C, R₉ --C, R_(9') --C, and R_(x) --C where "X" is 10 or greater. 17.The method of claim 10, wherein said derivative is selected from thegroup consisting of N--R₁ --C, N--R₂ --C, N--R₃ --C, N--R₄ --C, N--R₅--C, N--R₆ --C, N--R₇ --C, N--R₈ --C, N--R₉ --C, N--R_(9') --C, N--R_(x)--C where "X" is 10 or greater, and combinations thereof.
 18. The methodof claim 17, wherein said derivative is selected from the groupconsisting of N--R₁ --C, N--R₂ --C, N--R₃ --C, N--R₄ --C, N--R₅ --C,N--R₆ --C, N--R₇ --C, N--R₈ --C, N--R₉ --C, N--R_(9') --C, and N--R_(x)--C where "X" is 10 or greater.
 19. The method of claim 2, wherein saidcapsular polysaccharide is type-specific.
 20. The method of claim 8,wherein said capsular polysaccharide is type-specific.
 21. The method ofclaim 10, wherein said C protein alpha antigen derivative of saidconjugate vaccine is selected from the group consisting of N, C, N--C,R₁, R₂, R₃, R₄, R₅, R₆, R₇, R₈, R₉, R_(9'), R_(x), N--R₁, N--R₂, N--R₃,N--R₄, N--R₅, N--R₆, N--R₇, N--R₈, N--R₉, N--R_(9'), N--R_(x), R₁ --C,R₂ --C, R₃ --C, R₄ --C, R₅ --C, R₆ --C, R₇ --C, R₈ --C, R₉ --C, R_(9')--C, R_(x) --C, N--R₁ --C, N--R₂ --C, N--R₃ --C, N--R₄ --C, N--R₅ --C,N--R₆ --C, N--R₇ --C, N--R₈ --C, N--R₉ --C, N--R_(9') --C, and N--R_(x)--C where "X" is 10 or greater.